Amyloid precursor protein (APP) RNAi agent compositions and methods of use thereof

ABSTRACT

The disclosure relates to double stranded ribonucleic acid (dsRNAi) agents and compositions targeting the APP gene, as well as methods of inhibiting expression of an APP gene and methods of treating subjects having an APP-associated disease or disorder, such as cerebral amyloid angiopathy (CAA) and early onset familial Alzheimer disease (EOFAD or eFAD), using such dsRNAi agents and compositions.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of PCT Application No.PCT/US19/67449, filed Dec. 19, 2019, which claims the benefit of andpriority to U.S. Provisional Application No. 62/928,795, filed Oct. 31,2019, U.S. Provisional Application No. 62/862,472, filed Jun. 17, 2019,and U.S. Provisional Application No. 62/781,774, filed Dec. 19, 2018,the entire contents of which are hereby incorporated by reference.

FIELD OF THE INVENTION

The instant disclosure relates generally to APP-targeting RNAi agentsand methods.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been filedelectronically in ASCII format and is hereby incorporated by referencein its entirety. Said ASCII copy, created on Dec. 18, 2019, is named53433_500WO01_SequenceListing_ST25.txt and is 632 kB in size.

BACKGROUND OF THE INVENTION

The amyloid precursor protein (APP) gene encodes an integral membraneprotein expressed in neurons and glia. While the primary function of APPis unknown, secretase-cleaved forms of APP—particularly the AP cleavageforms of APP, e.g., Aβ(1-42) (aka Aβ42) and Aβ(1-40) (aka Aβ40) commonlyfound as the predominant protein in amyloid beta plaques—have long beendescribed as associated with the development and progression ofAlzheimer's disease (AD) in affected individuals. Indeed, identificationof myloid beta plaques in a subject is necessary for pathologicaldiagnosis of AD. Aβ cleavage forms of APP have been particularlydescribed to play a critical and even causal role in the development oftwo AD-related/associated diseases: cerebral amyloid angiopathy (CAA)and early onset familial Alzheimer disease (EOFAD or eFAD).

Inhibition of the expression and/or activity of APP with an agent thatcan selectively and efficiently inhibit APP, and thereby block or dampenthe production and/or levels of Aβ cleavage forms of APP, would beuseful for preventing or treating a variety of APP-associated diseasesand disorders, including AD, CAA and EOFAD, among others.

Current treatment options for APP-associated diseases and disorders areboth limited and largely ineffective. There are no existing therapiesfor hereditary CAA, and attempts to treat sporadic forms of AD and EOFADhave to date proven unsuccessful—for example, all trials of BACE1(β-secretase) inhibitors for treatment of sporadic AD have thus farfailed (Egan et al. The New England Journal of Medicine, 378: 1691-1703;Hung and Fu. Journal of Biomedical Science, 24: 47). Meanwhile, a numberof Aβ-directed immunotherapies are in various phases of development,while a number of human γ-secretase inhibitor programs have been haltedfor toxicity (Selkoe and Hardy. EMBO Molecular Medicine, 8: 595-608). Todate, approved pharmacologic treatments for APP-associated diseases ordisorders are directed to treatment of symptoms, not to prevention orcure, and such treatments are of limited efficacy, particularly asAPP-associated diseases or disorders advance in an affected individual.Therefore, there is a need for therapies for subjects suffering fromAPP-associated diseases and disorders, including a particular need fortherapies for subjects suffering from hereditary CAA and EOFAD.

BRIEF SUMMARY OF THE INVENTION

The present disclosure provides RNAi compositions which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an amyloid precursor protein (APP) gene. The APP gene maybe within a cell, e.g., a cell within a subject, such as a human. Thepresent disclosure also provides methods of using the RNAi compositionsof the disclosure for inhibiting the expression of an APP gene and/orfor treating a subject who would benefit from inhibiting or reducing theexpression of an APP gene, e.g., a subject suffering or prone tosuffering from an APP-associated disease, for example, cerebral amyloidangiopathy (CAA) or Alzheimer's disease (AD), e.g., early onset familialAlzheimer disease (EOFAD).

Accordingly, in one aspect, the instant disclosure provides a doublestranded ribonucleic acid (RNAi) agent for inhibiting expression of anamyloid precursor protein (APP) gene, where the RNAi agent includes asense strand and an antisense strand, and where the antisense strandincludes a region of complementarity which includes at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense sequences listed in any one of Tables 2A, 2B, 3,5A, 5B, 6, 9, 10-15, 16A, 16B, 26, and 30. In certain embodiments,thymine-to-uracil and/or uracil-to-thymine differences between aligned(compared) sequences are not counted as nucleotides that differ betweenthe aligned (compared) sequences.

Another aspect of the instant disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene, where the dsRNA agent includes a sense strand and an antisensestrand, where the sense strand includes at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thesense strand sequences presented in Tables 2A, 2B, 3, 5A, 5B, 6, 9,10-15, 16A, 16B, 26, and 30; and where the antisense strand includes atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom any one of antisense strand nucleotide sequences presented inTables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, 26, and 30.

In one embodiment, at least one of the sense strand and the antisensestrand of the double stranded RNAi agent includes one or more lipophilicmoieties conjugated to one or more internal nucleotide positions,optionally via a linker or carrier.

An additional aspect of the disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene, where the dsRNA agent includes a sense strand and an antisensestrand, where the sense strand includes at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NOs: 1-14, where a substitution of auracil for any thymine of SEQ ID NOs: 1-14 (when comparing alignedsequences) does not count as a difference that contributes to thediffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NOs: 1-14; and where the antisense strand includesat least 15 contiguous nucleotides differing by no more than 3nucleotides from any one of the nucleotide sequences of SEQ ID NOs:15-28, where a substitution of a uracil for any thymine of SEQ ID NOs:15-28 (when comparing aligned sequences) does not count as a differencethat contributes to the differing by no more than 3 nucleotides from anyone of the nucleotide sequences of SEQ ID NOs: 15-28, where at least oneof the sense strand and the antisense strand includes one or morelipophilic moieties conjugated to one or more internal nucleotidepositions, optionally via a linker or carrier.

In one embodiment, the double stranded RNAi agent sense strand includesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of the sense strand nucleotidesequence of an AD-392911, AD-392912, AD-392816, AD-392704, AD-392843,AD-392855, AD-392840, AD-392835, AD-392729, AD-392916, AD-392876,AD-392863, AD-392917, AD-392783, AD-392765, AD-392791, AD-392800,AD-392711, AD-392801, AD-392826, AD-392818, AD-392792, AD-392802,AD-392766, AD-392767, AD-392834, AD-392974, AD-392784, AD-392744,AD-392752, AD-392737, AD-392918, AD-392919, AD-392803, AD-392804,AD-392827, AD-392828, AD-392785, AD-392829, AD-392920, AD-392921,AD-392768, AD-392805, AD-392769, AD-392753, AD-392714, AD-392703,AD-392715, AD-392836, AD-392966, AD-392832, AD-392972, AD-392961,AD-392967, AD-392894, AD-392864, AD-392865, AD-392922, AD-392833,AD-392968, AD-392962, AD-392963, AD-392969, AD-392973, AD-392923,AD-392866, AD-392877, AD-392707, AD-392926, AD-392927, AD-392717,AD-392700, AD-392878, AD-392718, AD-392929, AD-392819, AD-392745,AD-392770, AD-392806, AD-392771, AD-392820, AD-392821, AD-392786,AD-392772, AD-392699, AD-392868, AD-392719, AD-392880, AD-392930,AD-392932, AD-392869, AD-392870, AD-392896, AD-392720, AD-392746,AD-392773, AD-392807, AD-392730, AD-392721, AD-392933, AD-392881,AD-392897, AD-392898, AD-392899, AD-392935, AD-392882, AD-392738,AD-392739, AD-392936, AD-392900, AD-392901, AD-392937, AD-392883,AD-392975, AD-392938, AD-392902, AD-392941, AD-392942, AD-392943,AD-392944, AD-392903, AD-392775, AD-392758, AD-392945, AD-392884,AD-392947, AD-392748, AD-392759, AD-392837, AD-392970, AD-392976,AD-392965, AD-392831, AD-392904, AD-392885, AD-392886, AD-392776,AD-392887, AD-392722, AD-392760, AD-392731, AD-392709, AD-392723,AD-392948, AD-392724, AD-392949, AD-392725, AD-392950, AD-392732,AD-392726, AD-392862, AD-392951, AD-392871, AD-392872, AD-397183,AD-397175, AD-397177, AD-397176, AD-397260, AD-397266, AD-397267,AD-397178, AD-397180, AD-397184, AD-397179, AD-397224, AD-397225,AD-397203, AD-397185, AD-397195, AD-397204, AD-397191, AD-397251,AD-397240, AD-397205, AD-397254, AD-397259, AD-397247, AD-397233,AD-397181, AD-397196, AD-397197, AD-397226, AD-397212, AD-397182,AD-397227, AD-397217, AD-397213, AD-397229, AD-397264, AD-397265,AD-397209, AD-397192, AD-397210, AD-397219, AD-397214, AD-397220,AD-397230, AD-397231, AD-397193, AD-397190, AD-397200, AD-397248,AD-397207, AD-397211, AD-397243, AD-397246, AD-397223, AD-397202,AD-397256, AD-397257, AD-397258, AD-397250, AD-397244, AD-454972,AD-454973, AD-454842, AD-454843, AD-454844, AD-994379, AD-961583,AD-961584, AD-961585, or AD-961586 duplex.

In another embodiment, the double stranded RNAi agent antisense strandincludes at least 15 contiguous nucleotides differing by no more than 3nucleotides from the antisense nucleotide sequence of an AD-392911,AD-392912, AD-392816, AD-392704, AD-392843, AD-392855, AD-392840,AD-392835, AD-392729, AD-392916, AD-392876, AD-392863, AD-392917,AD-392783, AD-392765, AD-392791, AD-392800, AD-392711, AD-392801,AD-392826, AD-392818, AD-392792, AD-392802, AD-392766, AD-392767,AD-392834, AD-392974, AD-392784, AD-392744, AD-392752, AD-392737,AD-392918, AD-392919, AD-392803, AD-392804, AD-392827, AD-392828,AD-392785, AD-392829, AD-392920, AD-392921, AD-392768, AD-392805,AD-392769, AD-392753, AD-392714, AD-392703, AD-392715, AD-392836,AD-392966, AD-392832, AD-392972, AD-392961, AD-392967, AD-392894,AD-392864, AD-392865, AD-392922, AD-392833, AD-392968, AD-392962,AD-392963, AD-392969, AD-392973, AD-392923, AD-392866, AD-392877,AD-392707, AD-392926, AD-392927, AD-392717, AD-392700, AD-392878,AD-392718, AD-392929, AD-392819, AD-392745, AD-392770, AD-392806,AD-392771, AD-392820, AD-392821, AD-392786, AD-392772, AD-392699,AD-392868, AD-392719, AD-392880, AD-392930, AD-392932, AD-392869,AD-392870, AD-392896, AD-392720, AD-392746, AD-392773, AD-392807,AD-392730, AD-392721, AD-392933, AD-392881, AD-392897, AD-392898,AD-392899, AD-392935, AD-392882, AD-392738, AD-392739, AD-392936,AD-392900, AD-392901, AD-392937, AD-392883, AD-392975, AD-392938,AD-392902, AD-392941, AD-392942, AD-392943, AD-392944, AD-392903,AD-392775, AD-392758, AD-392945, AD-392884, AD-392947, AD-392748,AD-392759, AD-392837, AD-392970, AD-392976, AD-392965, AD-392831,AD-392904, AD-392885, AD-392886, AD-392776, AD-392887, AD-392722,AD-392760, AD-392731, AD-392709, AD-392723, AD-392948, AD-392724,AD-392949, AD-392725, AD-392950, AD-392732, AD-392726, AD-392862,AD-392951, AD-392871, AD-392872, AD-397183, AD-397175, AD-397177,AD-397176, AD-397260, AD-397266, AD-397267, AD-397178, AD-397180,AD-397184, AD-397179, AD-397224, AD-397225, AD-397203, AD-397185,AD-397195, AD-397204, AD-397191, AD-397251, AD-397240, AD-397205,AD-397254, AD-397259, AD-397247, AD-397233, AD-397181, AD-397196,AD-397197, AD-397226, AD-397212, AD-397182, AD-397227, AD-397217,AD-397213, AD-397229, AD-397264, AD-397265, AD-397209, AD-397192,AD-397210, AD-397219, AD-397214, AD-397220, AD-397230, AD-397231,AD-397193, AD-397190, AD-397200, AD-397248, AD-397207, AD-397211,AD-397243, AD-397246, AD-397223, AD-397202, AD-397256, AD-397257,AD-397258, AD-397250, AD-397244, AD-454972, AD-454973, AD-454842,AD-454843, AD-454844, AD-994379, AD-961583, AD-961584, AD-961585, orAD-961586 duplex.

Optionally, the double stranded RNAi agent includes at least onemodified nucleotide.

In certain embodiments, the lipophilicity of the lipophilic moiety,measured by log K_(ow), exceeds 0.

In some embodiments, the hydrophobicity of the double-stranded RNAiagent, measured by the unbound fraction in a plasma protein bindingassay of the double-stranded RNAi agent, exceeds 0.2. In a relatedembodiment, the plasma protein binding assay is an electrophoreticmobility shift assay using human serum albumin protein.

In certain embodiments, all of the nucleotides of the sense strand aremodified nucleotides.

In some embodiments, substantially all of the nucleotides of theantisense strand are modified nucleotides. Optionally, all of thenucleotides of the sense strand are modified nucleotides.

In certain embodiments, all of the nucleotides of the antisense strandare modified nucleotides. Optionally, all of the nucleotides of thesense strand and all of the nucleotides of the antisense strand aremodified nucleotides.

In one embodiment, at least one of the modified nucleotides is adeoxy-nucleotide, a 3′-terminal deoxy-thymine (dT) nucleotide, a2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a2′-deoxy-modified nucleotide, a locked nucleotide, an unlockednucleotide, a conformationally restricted nucleotide, a constrainedethyl nucleotide, an abasic nucleotide, a 2′-amino-modified nucleotide,a 2′-O-allyl-modified nucleotide, 2′-C-alkyl-modified nucleotide,2′-hydroxly-modified nucleotide, a 2′-methoxyethyl modified nucleotide,a 2′-O-alkyl-modified nucleotide, a morpholino nucleotide, aphosphoramidate, a non-natural base comprising nucleotide, atetrahydropyran modified nucleotide, a 1,5-anhydrohexitol modifiednucleotide, a cyclohexenyl modified nucleotide, a nucleotide comprisinga 5′-phosphorothioate group, a nucleotide comprising a5′-methylphosphonate group, a nucleotide comprising a 5′ phosphate or 5′phosphate mimic, a nucleotide comprising vinyl phosphate, a nucleotidecomprising adenosine-glycol nucleic acid (GNA), a nucleotide comprisingthymidine-glycol nucleic acid (GNA)S-Isomer, a nucleotide comprising2-hydroxymethyl-tetrahydrofurane-5-phosphate, a nucleotide comprising2′-deoxythymidine-3′phosphate, a nucleotide comprising2′-deoxyguanosine-3′-phosphate, or a terminal nucleotide linked to acholesteryl derivative and/or a dodecanoic acid bisdecylamide group.

In a related embodiment, the modified nucleotide is a 2′-deoxy-2′-fluoromodified nucleotide, a 2′-deoxy-modified nucleotide, 3′-terminaldeoxy-thymine nucleotides (dT), a locked nucleotide, an abasicnucleotide, a 2′-amino-modified nucleotide, a 2′-alkyl-modifiednucleotide, a morpholino nucleotide, a phosphoramidate, and/or anon-natural base comprising nucleotide.

In one embodiment, the modified nucleotide includes a short sequence of3′-terminal deoxy-thymine nucleotides (dT).

In another embodiment, the modifications on the nucleotides are2′-O-methyl, 2′fluoro and GNA modifications.

In an additional embodiment, the double stranded RNAi agent includes atleast one phosphorothioate internucleotide linkage. Optionally, thedouble stranded RNAi agent includes 6-8 phosphorothioate internucleotidelinkages.

In certain embodiments, the region of complementarity is at least 17nucleotides in length. Optionally, the region of complementarity is19-23 nucleotides in length. Optionally, the region of complementarityis 19 nucleotides in length.

In one embodiment, each strand is no more than 30 nucleotides in length.

In another embodiment, at least one strand includes a 3′ overhang of atleast 1 nucleotide. Optionally, at least one strand includes a 3′overhang of at least 2 nucleotides.

In certain embodiments, the double stranded RNAi agent further includesa C16 ligand conjugated to the 3′ end, the 5′ end, or the 3′ end and the5′ end of the sense strand through a monovalent or branched bivalent ortrivalent linker.

In one embodiment, the ligand is

where B is a nucleotide base or a nucleotide base analog, optionallywhere B is adenine, guanine, cytosine, thymine or uracil.

In another embodiment, the region of complementarity includes any one ofthe antisense sequences in any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9,10-15, 16A, 16B, 26 and 30.

In an additional embodiment, the region of complementarity is that ofany one of the antisense sequences in any one of Tables 2A, 2B, 3, 5A,5B, 6, 9, 10-15, 16A, 16B, 26 and 30.

In some embodiments, the internal nucleotide positions include allpositions except the terminal two positions from each end of the strand.

In a related embodiment, the internal positions include all positionsexcept terminal three positions from each end of the strand. Optionally,the internal positions exclude the cleavage site region of the sensestrand.

In one embodiment, the internal positions exclude positions 9-12,counting from the 5′-end of the sense strand.

In another embodiment, the internal positions exclude positions 11-13,counting from the 3′-end of the sense strand. Optionally, the internalpositions exclude the cleavage site region of the antisense strand.

In one embodiment, the internal positions exclude positions 12-14,counting from the 5′-end of the antisense strand.

In another embodiment, the internal positions excluding positions 11-13on the sense strand, counting from the 3′-end, and positions 12-14 onthe antisense strand, counting from the 5′-end.

In an additional embodiment, one or more lipophilic moieties areconjugated to one or more of the following internal positions: positions4-8 and 13-18 on the sense strand, and positions 6-10 and 15-18 on theantisense strand, counting from the 5′end of each strand. Optionally,one or more lipophilic moieties are conjugated to one or more of thefollowing internal positions: positions 5, 6, 7, 15, and 17 on the sensestrand, and positions 15 and 17 on the antisense strand, counting fromthe 5′-end of each strand.

In certain embodiments, the lipophilic moiety is an aliphatic,alicyclic, or polyalicyclic compound. Optionally, the lipophilic moietyis lipid, cholesterol, retinoic acid, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine.

In some embodiments, the lipophilic moiety contains a saturated orunsaturated C₄-C₃₀ hydrocarbon chain, and an optional functional groupselected that is hydroxyl, amine, carboxylic acid, sulfonate, phosphate,thiol, azide, and/or alkyne.

In certain embodiments, the lipophilic moiety contains a saturated orunsaturated C₆-C₁₈ hydrocarbon chain. Optionally, the lipophilic moietycontains a saturated or unsaturated C₁₆ hydrocarbon chain. In a relatedembodiment, the lipophilic moiety is conjugated via a carrier thatreplaces one or more nucleotide(s) in the internal position(s). Incertain embodiments, the carrier is a cyclic group that is pyrrolidinyl,pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl, piperidinyl,piperazinyl, [1,3]dioxolanyl, oxazolidinyl, isoxazolidinyl, morpholinyl,thiazolidinyl, isothiazolidinyl, quinoxalinyl, pyridazinonyl,tetrahydrofuranyl, or decalinyl; or is an acyclic moiety based on aserinol backbone or a diethanolamine backbone.

In some embodiments, the lipophilic moiety is conjugated to thedouble-stranded RNAi agent via a linker containing an ether, thioether,urea, carbonate, amine, amide, maleimide-thioether, disulfide,phosphodiester, sulfonamide linkage, a product of a click reaction, orcarbamate.

In one embodiment, the lipophilic moiety is conjugated to a nucleobase,sugar moiety, or internucleosidic linkage.

In another embodiment, the double-stranded RNAi agent further includes aphosphate or phosphate mimic at the 5′-end of the antisense strand.Optionally, the phosphate mimic is a 5′-vinyl phosphonate (VP).

In certain embodiments, the double-stranded RNAi agent further includesa targeting ligand that targets a receptor which mediates delivery to aCNS tissue. In one embodiment, the targeting ligand is a C16 ligand.

In some embodiments, the double-stranded RNAi agent further includes atargeting ligand that targets a brain tissue.

In one embodiment, the lipophilic moeity or targeting ligand isconjugated via a bio-cleavable linker that is DNA, RNA, disulfide,amide, functionalized monosaccharides or oligosaccharides ofgalactosamine, glucosamine, glucose, galactose, mannose, and/or acombination thereof.

In a related embodiment, the 3′ end of the sense strand is protected viaan end cap which is a cyclic group having an amine, the cyclic groupbeing pyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl,imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolanyl, oxazolidinyl,isoxazolidinyl, morpholinyl, thiazolidinyl, isothiazolidinyl,quinoxalinyl, pyridazinonyl, tetrahydrofuranyl, or decalinyl.

In one embodiment, the RNAi agent includes at least one modifiednucleotide that is a 2′-O-methyl modified nucleotide, a 2′-fluoromodified nucleotide, a nucleotide that includes a glycol nucleic acid(GNA) and/or a nucleotide that includes a vinyl phosphate. Optionally,the RNAi agent includes at least one of each of the followingmodifications: 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a nucleotide comprising a glycol nucleic acid (GNA) and anucleotide comprising vinyl phosphate.

In another embodiment, the RNAi agent includes a pattern of modifiednucleotides as shown in FIG. 1A, FIG. 1B, Table 2A, Table 5A, or Table 9(where locations of 2′-C16, 2′-O-methyl, GNA, phosphorothioate and2′-fluoro modifications are as displayed in FIG. 1A, FIG. 1B, Table 2A,Table 5A, or Table 9, irrespective of the individual nucleotide basesequences of the displayed RNAi agents).

Another aspect of the instant disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene, where the double stranded RNAi agent includes a sense strandcomplementary to an antisense strand, where the antisense strandincludes a region complementary to part of an mRNA encoding APP, whereeach strand is about 14 to about 30 nucleotides in length, where thedouble stranded RNAi agent is represented by formula (III):sense: 5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′antisense: 3′n _(p)′-N _(a)′-(X′X′X′)_(k)-N _(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N _(a)′-n _(q)′5′  (III)where:j, k, and 1 are each independently 0 or 1;p, p′, q, and q′ are each independently 0-6;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence including 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence including at least twodifferently modified nucleotides;each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 0-10 nucleotides which are either modified orunmodified or combinations thereof;each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent, independently represents an overhang nucleotide;XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; andwhere the sense strand is conjugated to at least one ligand.

-   -   In one embodiment, i is 0; j is 0; i is 1; j is 1; both i and j        are 0; or both i and j are 1.    -   In another embodiment, k is 0; l is 0; k is 1; l is 1; both k        and 1 are 0; or both k and 1 are 1.    -   In certain embodiments, XXX is complementary to X′X′X′, YYY is        complementary to Y′Y′Y′, and ZZZ is complementary to Z′Z′Z′.    -   In another embodiment, the YYY motif occurs at or near the        cleavage site of the sense strand.    -   In an additional embodiment, the Y′Y′Y′ motif occurs at the 11,        12 and 13 positions of the antisense strand from the 5′-end.        Optionally, the Y′ is 2′-O-methyl.    -   In some embodiments, formula (III) is represented by formula        (Ma):        sense: 5′n _(p)-N _(a)-YYY-N _(a)-n _(q)3′        antisense: 3′n _(p′)-N _(a′)-Y′Y′Y′-N _(a′)-n _(q′)5′  (IIIa).

In another embodiment, formula (III) is represented by formula (IIIb):sense: 5′n _(p)-N _(a)-YYY-N _(b)-ZZZ-N _(a)-n _(q)3′antisense: 3′n _(p′)-N _(a′)-Y′Y′Y′-N _(b′)-Z′Z′Z′-N _(a′)-n_(q′)5′  (IIIb)where each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 1-5 modified nucleotides.

In an additional embodiment, formula (III) is represented by formula(IIIc):sense: 5′n _(p)-N _(a)-XXX-N _(b)-YYY-N _(a)-n _(q)3′antisense: 3′n _(p′)-N _(a′)-X′X′X′-N _(b′)-Y′Y′Y′-N _(a)′-n_(q′)5′  (IIIc)where each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 1-5 modified nucleotides.

In certain embodiments, formula (III) is represented by formula (IIId):sense: 5′n _(p)-N _(a)-XXX-N _(b)-YYY-N _(b)-ZZZ-N _(a)-n _(q)3′antisense: 3′n _(p)′-N _(a)′-X′X′X′-N _(b′)-Y′Y′Y′-N _(b′)-Z′Z′Z′-N_(a′)-n _(q′)5′  (IIId)where each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 1-5 modified nucleotides and each N_(a) and N_(a)′independently represents an oligonucleotide sequence including 2-10modified nucleotides.

In another embodiment, the double stranded region is 15-30 nucleotidepairs in length. Optionally, the double stranded region is 17-23nucleotide pairs in length.

In certain embodiments, the double stranded region is 17-25 nucleotidepairs in length. Optionally, the double stranded region is 23-27nucleotide pairs in length.

In some embodiments, the double stranded region is 19-21 nucleotidepairs in length. Optionally, the double stranded region is 21-23nucleotide pairs in length.

In certain embodiments, each strand has 15-30 nucleotides. Optionally,each strand has 19-30 nucleotides.

In another embodiment, the modifications on the nucleotides of the RNAiagent are LNA, glycol nucleic acid (GNA), HNA, CeNA, 2′-methoxyethyl,2′-O-alkyl, 2′-O-allyl, 2′-C-allyl, 2′-fluoro, 2′-deoxy and/or2′-hydroxyl, and combinations thereof. Optionally, the modifications onnucleotides include 2′-O-methyl, 2′-fluoro and/or GNA, and combinationsthereof. In a related embodiment, the modifications on the nucleotidesare 2′-O-methyl or 2′-fluoro modifications.

In one embodiment the RNAi agent includes a ligand that is or includesone or more C16 moieties attached through a bivalent or trivalentbranched linker.

In certain embodiments, the ligand is attached to the 3′ end of thesense strand.

In some embodiments, the RNAi agent further includes at least onephosphorothioate or methylphosphonate internucleotide linkage. In arelated embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the 3′-terminus of one strand. Optionally,the strand is the antisense strand. In another embodiment, the strand isthe sense strand. In a related embodiment, the phosphorothioate ormethylphosphonate internucleotide linkage is at the 5′-terminus of onestrand. Optionally, the strand is the antisense strand. In anotherembodiment, the strand is the sense strand.

In another embodiment, the phosphorothioate or methylphosphonateinternucleotide linkage is at the both the 5′- and 3′-terminus of onestrand. Optionally, the strand is the antisense strand. In anotherembodiment, the strand is the sense strand.

In an additional embodiment, the base pair at the 1 position of the5′-end of the antisense strand of the RNAi agent duplex is an A:U basepair.

In certain embodiments, the Y nucleotides contain a 2′-fluoromodification.

In some embodiments, the Y′ nucleotides contain a 2′-O-methylmodification.

In certain embodiments, p′>0. Optionally, p′=2.

In some embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides arecomplementary to the target mRNA.

In certain embodiments, q′=0, p=0, q=0, and p′ overhang nucleotides arenon-complementary to the target mRNA.

In one embodiment, the sense strand of the RNAi agent has a total of 21nucleotides and the antisense strand has a total of 23 nucleotides.

In another embodiment, at least one n_(p)′ is linked to a neighboringnucleotide via a phosphorothioate linkage. Optionally, all n_(p)′ arelinked to neighboring nucleotides via phosphorothioate linkages.

In certain embodiments, the RNAi agent of the instant disclosure is oneof those listed in Table 2A, 2B, 3, 5A, 5B, 6 and/or 9.

In some embodiments, all of the nucleotides of the sense strand and allof the nucleotides of the antisense strand include a modification.

Another aspect of the instant disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene in a cell, where the double stranded RNAi agent includes a sensestrand complementary to an antisense strand, where the antisense strandincludes a region complementary to part of an mRNA encoding APP, whereeach strand is about 14 to about 30 nucleotides in length, where thedouble stranded RNAi agent is represented by formula (III):sense: 5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′antisense: 3′n _(p)′-N _(a)′-(X′X′X′)_(k)-N _(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N _(a)′-n _(q)′5′  (III)where:j, k, and 1 are each independently 0 or 1;p, p′, q, and q′ are each independently 0-6;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence including 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence including at least twodifferently modified nucleotides;each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 0-10 nucleotides which are either modified orunmodified or combinations thereof;each n_(p), n_(p)′, n_(q), and n_(q)′, each of which may or may not bepresent independently represents an overhang nucleotide;XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and where the modifications are 2′-O-methyl or 2′-fluoromodifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′; andwhere the sense strand is conjugated to at least one ligand.

An additional aspect of the instant disclosure provides a doublestranded RNAi agent for inhibiting expression of an amyloid precursorprotein (APP) gene in a cell, where the double stranded RNAi agentincludes a sense strand complementary to an antisense strand, where theantisense strand includes a region complementary to part of an mRNAencoding APP, where each strand is about 14 to about 30 nucleotides inlength, where the double stranded RNAi agent is represented by formula(III):sense: 5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′antisense: 3′n _(p)′-N _(a)′-(X′X′X′)_(k)-N _(b)′-Z′Z′Z′)_(l)-N _(a)′-n_(q)′5′  (III)where:i, j, k, and 1 are each independently 0 or 1;each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;p, q, and q′ are each independently 0-6;n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence including 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence including at least twodifferently modified nucleotides;each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 0-10 nucleotides which are either modified orunmodified or combinations thereof;XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and where the modifications are 2′-O-methyl, glycol nucleicacid (GNA) or 2′-fluoro modifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; andwhere the sense strand is conjugated to at least one ligand.

Another aspect of the instant disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene in a cell, where the double stranded RNAi agent includes a sensestrand complementary to an antisense strand, where the antisense strandincludes a region complementary to part of an mRNA encoding APP, whereeach strand is about 14 to about 30 nucleotides in length, where thedouble stranded RNAi agent is represented by formula (III):sense: 5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′antisense: 3′n _(p)′-N _(a)′-(X′X′X′)_(k)-N _(b) ′Z′Z′Z′)_(l)-N _(a)′-n_(q)′5′  (III)where:i, j, k, and 1 are each independently 0 or 1;each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;p, q, and q′ are each independently 0-6;n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence including 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence including at least twodifferently modified nucleotides;each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 0-10 nucleotides which are either modified orunmodified or combinations thereof;XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and where the modifications are 2′-O-methyl or 2′-fluoromodifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y; andwhere the sense strand is conjugated to at least one ligand, optionallywhere the ligand is one or more C16 ligands.

An additional aspect of the instant disclosure provides a doublestranded RNAi agent for inhibiting expression of an amyloid precursorprotein (APP) gene in a cell, where the double stranded RNAi agentincludes a sense strand complementary to an antisense strand, where theantisense strand includes a region complementary to part of an mRNAencoding APP, where each strand is about 14 to about 30 nucleotides inlength, where the double stranded RNAi agent is represented by formula(III):sense: 5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′antisense: 3′n _(p)′-N _(a)′-(X′X′X′)_(k)-N _(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N _(a)′-n _(q)′5′  (III)where:j, k, and 1 are each independently 0 or 1;each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;p, q, and q′ are each independently 0-6;n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence including 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence including at least twodifferently modified nucleotides;each N_(b) and N_(b)′ independently represents an oligonucleotidesequence including 0-10 nucleotides which are either modified orunmodified or combinations thereof;XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides, and where the modifications are 2′-O-methyl or 2′-fluoromodifications;modifications on N_(b) differ from the modification on Y andmodifications on N_(b)′ differ from the modification on Y′;where the sense strand includes at least one phosphorothioate linkage;andwhere the sense strand is conjugated to at least one ligand, optionallywhere the ligand is one or more C16 ligands.

Another aspect of the instant disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene in a cell, where the double stranded RNAi agent includes a sensestrand complementary to an antisense strand, where the antisense strandincludes a region complementary to part of an mRNA encoding APP, whereeach strand is about 14 to about 30 nucleotides in length, where thedouble stranded RNAi agent is represented by formula (III):sense: 5′n _(p)-N _(a)-YYY-N _(a)-n _(q)3′antisense: 3′n _(p)′-N _(a)′-Y′Y′Y′-N _(a)′-n _(q)′5′  (IIIa)where:each n_(p), n_(q), and n_(q)′, each of which may or may not be present,independently represents an overhang nucleotide;p, q, and q′ are each independently 0-6;n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia a phosphorothioate linkage;each N_(a) and N_(a)′ independently represents an oligonucleotidesequence including 0-25 nucleotides which are either modified orunmodified or combinations thereof, each sequence including at least twodifferently modified nucleotides;YYY and Y′Y′Y′ each independently represent one motif of three identicalmodifications on three consecutive nucleotides, and where themodifications are 2′-O-methyl or 2′-fluoro modifications;where the sense strand includes at least one phosphorothioate linkage;andwhere the sense strand is conjugated to at least one ligand, optionallywhere the ligand is one or more C16 ligands.

An additional aspect of the instant disclosure provides a doublestranded RNAi agent for inhibiting expression of an amyloid precursorprotein (APP) gene, where the double stranded RNAi agent includes asense strand and an antisense strand forming a double stranded region,where the sense strand includes at least 15 contiguous nucleotidesdiffering by no more than 3 nucleotides from any one of the nucleotidesequences of SEQ ID NOs: 1-14 and the antisense strand includes at least15 contiguous nucleotides differing by no more than 3 nucleotides fromany one of the nucleotide sequences of SEQ ID NOs: 15-28, wheresubstantially all of the nucleotides of the sense strand include amodification that is a 2′-O-methyl modification, a GNA and/or a2′-fluoro modification, where the sense strand includes twophosphorothioate internucleotide linkages at the 5′-terminus, wheresubstantially all of the nucleotides of the antisense strand include amodification selected from the group consisting of a 2′-O-methylmodification and a 2′-fluoro modification, where the antisense strandincludes two phosphorothioate internucleotide linkages at the5′-terminus and two phosphorothioate internucleotide linkages at the3′-terminus, and where the sense strand is conjugated to one or more C16ligands.

Another aspect of the instant disclosure provides a double stranded RNAiagent for inhibiting expression of an amyloid precursor protein (APP)gene, where the double stranded RNAi agent includes a sense strand andan antisense strand forming a double stranded region, where the sensestrand includes at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from any one of the nucleotide sequences of SEQ IDNOs: 1-14 and the antisense strand includes at least 15 contiguousnucleotides differing by no more than 3 nucleotides from any one of thenucleotide sequences of SEQ ID NOs: 15-28, where the sense strandincludes at least one 3′-terminal deoxy-thymine nucleotide (dT), andwhere the antisense strand includes at least one 3′-terminaldeoxy-thymine nucleotide (dT).

In one embodiment, all of the nucleotides of the sense strand and all ofthe nucleotides of the antisense strand are modified nucleotides.

In another embodiment, each strand has 19-30 nucleotides.

In certain embodiments, the antisense strand of the RNAi agent includesat least one thermally destabilizing modification of the duplex withinthe first 9 nucleotide positions of the 5′ region or a precursorthereof. Optionally, the thermally destabilizing modification of theduplex is one or more of

where B is nucleobase.

Another aspect of the instant disclosure provides a cell containing adouble stranded RNAi agent of the instant disclosure.

An additional aspect of the instant disclosure provides a pharmaceuticalcomposition for inhibiting expression of an APP gene that includes adouble stranded RNAi agent of the instant disclosure.

In one embodiment, the double stranded RNAi agent is administered in anunbuffered solution. Optionally, the unbuffered solution is saline orwater.

In another embodiment, the double stranded RNAi agent is administeredwith a buffer solution. Optionally, the buffer solution includesacetate, citrate, prolamine, carbonate, or phosphate or any combinationthereof. In another embodiment, the buffer solution is phosphatebuffered saline (PBS).

Another aspect of the disclosure provides a pharmaceutical compositionthat includes a double stranded RNAi agent of the instant disclosure anda lipid formulation.

In one embodiment, the lipid formulation includes a LNP.

An additional aspect of the disclosure provides a method of inhibitingexpression of an amyloid precursor protein (APP) gene in a cell, themethod involving: (a)

contacting the cell with a double stranded RNAi agent of the instantdisclosure or a pharmaceutical composition of of the instant disclosure;and (b) maintaining the cell produced in step (a) for a time sufficientto obtain degradation of the mRNA transcript of an APP gene, therebyinhibiting expression of the APP gene in the cell.

In one embodiment, the cell is within a subject. Optionally, the subjectis a human.

In certain embodiments, the subject is a rhesus monkey, a cynomolgousmonkey, a mouse, or a rat.

In one embodiment, the human subject suffers from an APP-associateddisorder. Optionally, the APP-associated disease is cerebral amyloidangiopathy (CAA).

In another embodiment, the APP-associated disorder is early onsetfamilial Alzheimer disease (EOFAD). In an additional embodiment, theAPP-associated disorder is Alzheimer's disease (AD).

In certain embodiments APP expression is inhibited by at least about 30%by the RNAi agent.

Another aspect of the disclosure provides a method of treating a subjecthaving a disorder that would benefit from a reduction in APP expression,the method involving administering to the subject a therapeuticallyeffective amount of a double stranded RNAi agent of the disclosure or apharmaceutical composition of the disclosure, thereby treating thesubject.

In certain embodiments, the method further involves administering anadditional therapeutic agent to the subject.

In certain embodiments, the double stranded RNAi agent is administeredat a dose of about 0.01 mg/kg to about 50 mg/kg.

In some embodiments, the double stranded RNAi agent is administered tothe subject intrathecally.

In certain embodiments, the administration of the double stranded RNAito the subject causes a decrease in Aβ accumulation. Optionally, theadministration of the double stranded RNAi to the subject causes adecrease in Aβ(1-40) and/or Aβ(1-42) accumulation.

In related embodiments, the administration of the dsRNA to the subjectcauses a decrease in amyloid plaque formation and/or accumulation in thesubject.

In one embodiment, the method reduces the expression of a target gene ina brain or spine tissue. Optionally, the brain or spine tissue iscortex, cerebellum, striatum, cervical spine, lumbar spine, and/orthoracic spine.

Another aspect of the instant disclosure provides a method of inhibitingthe expression of APP in a subject, the method involving: administeringto the subject a therapeutically effective amount of a double strandedRNAi agent of the disclosure or a pharmaceutical composition of thedisclosure, thereby inhibiting the expression of APP in the subject.

An additional aspect of the disclosure provides a method for treating orpreventing an APP-associated disease or disorder in a subject, themethod involving administering to the subject a therapeuticallyeffective amount of a double stranded RNAi agent of the disclosure or apharmaceutical composition of the disclosure, thereby treating orpreventing an APP-associated disease or disorder in the subject.

In certain embodiments, the APP-associated disease or disorder iscerebral amyloid angiopathy (CAA) and/or Alzheimer's disease (AD).Optionally, the AD is early onset familial Alzheimer disease (EOFAD).

Another aspect of the instant disclosure provides a kit for performing amethod of the instant disclosure, the kit including: a) a doublestranded RNAi agent of the instant disclosure, and b) instructions foruse, and c) optionally, a means for administering the double strandedRNAi agent to the subject.

An additional aspect of the instant disclosure provides a doublestranded ribonucleic acid (RNAi) agent for inhibiting expression of anamyloid precursor protein (APP) gene, where the RNAi agent possesses asense strand and an antisense strand, and where the antisense strandincludes a region of complementarity which includes at least 15contiguous nucleotides differing by no more than 3 nucleotides from anyone of the antisense strand nucleobase sequences of AD-392911,AD-392912, AD-392816, AD-392704, AD-392843, AD-392855, AD-392840,AD-392835, AD-392729, AD-392916, AD-392876, AD-392863, AD-392917,AD-392783, AD-392765, AD-392791, AD-392800, AD-392711, AD-392801,AD-392826, AD-392818, AD-392792, AD-392802, AD-392766, AD-392767,AD-392834, AD-392974, AD-392784, AD-392744, AD-392752, AD-392737,AD-392918, AD-392919, AD-392803, AD-392804, AD-392827, AD-392828,AD-392785, AD-392829, AD-392920, AD-392921, AD-392768, AD-392805,AD-392769, AD-392753, AD-392714, AD-392703, AD-392715, AD-392836,AD-392966, AD-392832, AD-392972, AD-392961, AD-392967, AD-392894,AD-392864, AD-392865, AD-392922, AD-392833, AD-392968, AD-392962,AD-392963, AD-392969, AD-392973, AD-392923, AD-392866, AD-392877,AD-392707, AD-392926, AD-392927, AD-392717, AD-392700, AD-392878,AD-392718, AD-392929, AD-392819, AD-392745, AD-392770, AD-392806,AD-392771, AD-392820, AD-392821, AD-392786, AD-392772, AD-392699,AD-392868, AD-392719, AD-392880, AD-392930, AD-392932, AD-392869,AD-392870, AD-392896, AD-392720, AD-392746, AD-392773, AD-392807,AD-392730, AD-392721, AD-392933, AD-392881, AD-392897, AD-392898,AD-392899, AD-392935, AD-392882, AD-392738, AD-392739, AD-392936,AD-392900, AD-392901, AD-392937, AD-392883, AD-392975, AD-392938,AD-392902, AD-392941, AD-392942, AD-392943, AD-392944, AD-392903,AD-392775, AD-392758, AD-392945, AD-392884, AD-392947, AD-392748,AD-392759, AD-392837, AD-392970, AD-392976, AD-392965, AD-392831,AD-392904, AD-392885, AD-392886, AD-392776, AD-392887, AD-392722,AD-392760, AD-392731, AD-392709, AD-392723, AD-392948, AD-392724,AD-392949, AD-392725, AD-392950, AD-392732, AD-392726, AD-392862,AD-392951, AD-392871, AD-392872, AD-397183, AD-397175, AD-397177,AD-397176, AD-397260, AD-397266, AD-397267, AD-397178, AD-397180,AD-397184, AD-397179, AD-397224, AD-397225, AD-397203, AD-397185,AD-397195, AD-397204, AD-397191, AD-397251, AD-397240, AD-397205,AD-397254, AD-397259, AD-397247, AD-397233, AD-397181, AD-397196,AD-397197, AD-397226, AD-397212, AD-397182, AD-397227, AD-397217,AD-397213, AD-397229, AD-397264, AD-397265, AD-397209, AD-397192,AD-397210, AD-397219, AD-397214, AD-397220, AD-397230, AD-397231,AD-397193, AD-397190, AD-397200, AD-397248, AD-397207, AD-397211,AD-397243, AD-397246, AD-397223, AD-397202, AD-397256, AD-397257,AD-397258, AD-397250, AD-397244 AD-454972, AD-454973, AD-454842,AD-454843, AD-454844, AD-994379, AD-961583, AD-961584, AD-961585, orAD-961586.

In one embodiment, the RNAi agent includes one or more of the followingmodifications: a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-C-alkyl-modified nucleotide, a nucleotide comprising aglycol nucleic acid (GNA), a phosphorothioate (PS) and a vinylphosphonate (VP). Optionally, the RNAi agent includes at least one ofeach of the following modifications: a 2′-O-methyl modified nucleotide,a 2′-fluoro modified nucleotide, a 2′-C-alkyl-modified nucleotide, anucleotide comprising a glycol nucleic acid (GNA), a phosphorothioateand a vinyl phosphonate (VP).

In another embodiment, the RNAi agent includes four or more PSmodifications, optionally six to ten PS modifications, optionally eightPS modifications.

In an additional embodiment, each of the sense strand and the antisensestrand of the RNAi agent possesses a 5′-terminus and a 3′-terminus, andthe RNAi agent includes eight PS modifications positioned at each of thepenultimate and ultimate internucleotide linkages from the respective3′- and 5′-termini of each of the sense and antisense strands of theRNAi agent.

In another embodiment, each of the sense strand and the antisense strandof the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAiagent includes only one nucleotide including a GNA. Optionally, thenucleotide including a GNA is positioned on the antisense strand at theseventh nucleobase residue from the 5′-terminus of the antisense strand.

In an additional embodiment, each of the sense strand and the antisensestrand of the RNAi agent includes a 5′-terminus and a 3′-terminus, andthe RNAi agent includes between one and four 2′-C-alkyl-modifiednucleotides. Optionally, the 2′-C-alkyl-modified nucleotide is a2′-C16-modified nucleotide. Optionally, the RNAi agent includes a single2′-C16-modified nucleotide. Optionally, the single 2′-C16-modifiednucleotide is located on the sense strand at the sixth nucleobaseposition from the 5′-terminus of the sense strand or on the terminalnucleobase position of the 5′ end.

In another embodiment, each of the sense strand and the antisense strandof the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAiagent includes two or more 2′-fluoro modified nucleotides. Optionally,each of the sense strand and the antisense strand of the RNAi agentincludes two or more 2′-fluoro modified nucleotides. Optionally, the2′-fluoro modified nucleotides are located on the sense strand atnucleobase positions 7, 9, 10 and 11 from the 5′-terminus of the sensestrand and on the antisense strand at nucleobase positions 2, 14 and 16from the 5′-terminus of the antisense strand.

In an additional embodiment, each of the sense strand and the antisensestrand of the RNAi agent includes a 5′-terminus and a 3′-terminus, andthe RNAi agent includes one or more VP modifications. Optionally, theRNAi agent includes a single VP modification at the 5′-terminus of theantisense strand.

In another embodiment, each of the sense strand and the antisense strandof the RNAi agent includes a 5′-terminus and a 3′-terminus, and the RNAiagent includes two or more 2′-O-methyl modified nucleotides. Optionally,the RNAi agent includes 2′-O-methyl modified nucleotides at allnucleobase locations not modified by a 2′-fluoro, a 2′-C-alkyl or aglycol nucleic acid (GNA). Optionally, the two or more 2′-O-methylmodified nucleotides are located on the sense strand at positions 1, 2,3, 4, 5, 8, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 from the5′-terminus of the sense strand and on the antisense strand at positions1, 3, 4, 5, 6, 8, 9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22 and 23from the 5′-terminus of the antisense strand.

Another aspect of the instant disclosure provides a double strandedribonucleic acid (RNAi) agent for inhibiting expression of an amyloidprecursor protein (APP) gene, where the RNAi agent includes a sensestrand and an antisense strand, and where the antisense strand includesa region of at least 15 contiguous nucleobases in length that issufficiently complementary to a target APP sequence of APP NM 00484positions 1891-1919; APP NM_00484 positions 2282-2306; APP NM_00484positions 2464-2494; APP NM_00484 positions 2475-2638; APP NM_00484positions 2621-2689; APP NM_00484 positions 2682-2725; APP NM_00484positions 2705-2746; APP NM_00484 positions 2726-2771; APP NM_00484positions 2754-2788; APP NM_00484 positions 2782-2813; APP NM_00484positions 2801-2826; APP NM_00484 positions 2847-2890; APP NM_00484positions 2871-2896; APP NM_00484 positions 2882-2960; APP NM_00484positions 2942-2971; APP NM_00484 positions 2951-3057; APP NM_00484positions 3172-3223; APP NM_00484 positions 3209-3235; NM_00484positions 3256-3289; NM_00484 positions 3302-3338; APP NM_00484positions 3318-3353; APP NM_00484 positions 3334-3361, APPNM_001198823.1 positions 251-284; APP NM_001198823.1 positions 362-404;APP NM_001198823.1 positions 471-510; APP NM_001198823.1 positions532-587; APP NM_001198823.1 positions 601-649; APP NM_001198823.1positions 633-662; APP NM_001198823.1 positions 1351-1388; APPNM_001198823.1 positions 1609-1649; APP NM_001198823.1 positions1675-1698; APP NM_001198823.1 positions 1752-1787; APP NM_001198823.1positions 2165-2217; APP NM_001198823.1 positions 2280-2344; or APPNM_001198823.1 positions 2403-2431 to effect APP knockdown and thatdiffers by no more than 3 nucleotides across the at least 15 contiguousnucleobases sufficiently complementary to the APP target sequence toeffect APP knockdown.

Another aspect of the instant disclosure provides a double stranded RNAiagent that includes one or more modifications selected from the groupconsisting of a 2′-O-methyl modified nucleotide, a 2′-fluoro modifiednucleotide, a 2′-C-alkyl-modified nucleotide, a nucleotide comprising aglycol nucleic acid (GNA), a phosphorothioate (PS) and a vinylphosphonate (VP), optionally wherein said RNAi agent comprises at leastone of each modification selected from the group consisting of a2′-O-methyl modified nucleotide, a 2′-fluoro modified nucleotide, a2′-C-alkyl-modified nucleotide, a nucleotide comprising a glycol nucleicacid (GNA), a phosphorothioate and a vinyl phosphonate (VP).

Another aspect of the instant disclosure provides that the RNAi agentcomprises four or more PS modifications, optionally six to ten PSmodifications, optionally eight PS modifications.

Another aspect of the instant disclosure provides that each of the sensestrand and the antisense strand of the RNAi agent comprises a5′-terminus and a 3′-terminus, and wherein the RNAi agent compriseseight PS modifications positioned at the penultimate and ultimateinternucleotide linkages from the respective 3′- and 5′-termini of eachof the sense and antisense strands of the RNAi agent.

Another aspect of the instant disclosure provides that each of the sensestrand and the antisense strand of the RNAi agent comprises a5′-terminus and a 3′-terminus, and wherein the RNAi agent comprises onlyone nucleotide comprising a GNA, optionally wherein the nucleotidecomprising a GNA is positioned on the antisense strand at the seventhnucleobase residue from the 5′-terminus of the antisense strand.

Another aspect of the instant disclosure provides that each of the sensestrand and the antisense strand of the RNAi agent comprises a5′-terminus and a 3′-terminus, and wherein the RNAi agent comprisesbetween one and four 2′-C-alkyl-modified nucleotides, optionally whereinthe 2′-C-alkyl-modified nucleotide is a 2′-C16-modified nucleotide,optionally wherein the RNAi agent comprises a single 2′-C16-modifiednucleotide, optionally the single 2′-C16-modified nucleotide is locatedon the sense strand at the sixth nucleobase position from the5′-terminus of the sense strand or on the terminal nucleobase positionof the 5′ end.

Another aspect of the instant disclosure provides that each of the sensestrand and the antisense strand of the RNAi agent comprises a5′-terminus and a 3′-terminus, and wherein the RNAi agent comprises twoor more 2′-fluoro modified nucleotides, optionally wherein each of thesense strand and the antisense strand of the RNAi agent comprises two ormore 2′-fluoro modified nucleotides, optionally wherein the 2′-fluoromodified nucleotides are located on the sense strand at nucleobasepositions 7, 9, 10 and 11 from the 5′-terminus of the sense strand andon the antisense strand at nucleobase positions 2, 14 and 16 from the5′-terminus of the antisense strand.

Another aspect of the instant disclosure provides that each of the sensestrand and the antisense strand of the RNAi agent comprises a5′-terminus and a 3′-terminus, and wherein the RNAi agent comprises oneor more VP modifications, optionally wherein the RNAi agent comprises asingle VP modification at the 5′-terminus of the antisense strand.

Another aspect of the instant disclosure provides that each of the sensestrand and the antisense strand of the RNAi agent comprises a5′-terminus and a 3′-terminus, and wherein the RNAi agent comprises twoor more 2′-O-methyl modified nucleotides, optionally wherein the RNAiagent comprises 2′-O-methyl modified nucleotides at all nucleobaselocations not modified by a 2′-fluoro, a 2′-C-alkyl or a glycol nucleicacid (GNA), optionally wherein the two or more 2′-O-methyl modifiednucleotides are located on the sense strand at positions 1, 2, 3, 4, 5,8, 12, 13, 14, 15, 16, 17, 18, 19, 20 and 21 from the 5′-terminus of thesense strand and on the antisense strand at positions 1, 3, 4, 5, 6, 8,9, 10, 11, 12, 13, 15, 17, 18, 19, 20, 21, 22 and 23 from the5′-terminus of the antisense strand.

BRIEF DESCRIPTION OF THE DRAWINGS

The following detailed description, given by way of example, but notintended to limit the disclosure solely to the specific embodimentsdescribed, may best be understood in conjunction with the accompanyingdrawings, in which:

FIG. 1A and FIG. 1B show a schematic image of modified RNAi agentstested for in vivo hsAPP knockdown activity. The tested agents includeAD-392911 (sense strand SEQ ID NO:867, antisense strand SEQ ID NO:868),AD-392912 (sense strand SEQ ID NO:869; antisense strand SEQ ID NO:870),AD-392913 (sense strand SEQ ID NO:949; antisense strand SEQ ID NO:950),AD-392843 (sense strand SEQ ID NO:987; antisense strand SEQ ID NO:988),AD-392844 (sense strand SEQ ID NO:919; antisense strand SEQ ID NO:920),AD-392824 (sense strand SEQ ID NO:943; antisense strand SEQ ID NO:944),AD-392704 (sense strand SEQ ID NO:967; antisense strand SEQ ID NO:968),AD-392790 (sense strand SEQ ID NO:931; antisense strand SEQ ID NO:932),AD-392703 (sense strand SEQ ID NO:1079; antisense strand SEQ IDNO:1080), AD-392866 (sense strand SEQ ID NO:1087; antisense strand SEQID NO:1088), AD-392927 (sense strand SEQ ID NO:1141; antisense strandSEQ ID NO:1142), AD-392916 (sense strand SEQ ID NO:997; antisense strandSEQ ID NO:998), AD-392714 (sense strand SEQ ID NO:1077; antisense strandSEQ ID NO:1078), and AD-392926 (sense strand SEQ ID NO:1139; antisensestrand SEQ ID NO:1140).

FIG. 2A and FIG. 2B show in vivo hsAPP knockdown activity resultsobserved for the modified RNAi agents shown in FIG. 1A and FIG. 1B.

FIG. 3A is a scheme demonstrating the strategy to identify potent humanAPP (hAPP) siRNAs in targeting hereditary cerebral amyloid angiopathy(hCAA).

FIG. 3B is a plot of percent remaining mRNA in an in vitro endogenousscreen of hAPP siRNAs at a concentration of 10 nM in Be(2)C cells.

FIG. 4A is a scheme demonstrating the timing of APP siRNA transfectionin BE(2)C neuronal cells. APP siRNA was transfected at 10, 1, and 0.1 nMand assessed 24 and 48 hours after transfection.

FIG. 4B is a graph showing the applied concentration of APP duplex siRNAvs the percent remaining mRNA in BE(2)C cells 48 hours aftertransfection.

FIG. 4C is two graphs of soluble APP alpha (top) and beta (bottom)species in BE(2)C cells supernatant 48 hours after transfection.

FIG. 5A is a scheme demonstrating the APP siRNA non-human primate (NHP)screening study design. 5 compounds were assessed, and 5 animals wereused for each experiment. A single intrathecal (IT) injection of 72 mgof the compound of interest was given at the onset.

FIG. 5B is two graphs of soluble APP alpha (top) and beta (bottom)species in BE(2)C (bottom), post IT administration in cyno monkeys of 72mg of AD-454972 targeting APP.

FIG. 5C is a graph showing the results of tissue mRNA knockdown at day29 post IT administration in cyno monkeys of 72 mg of AD-454972targeting APP.

FIG. 5D is a scheme demonstrating the structure of the AD-454972 (sensestrand SEQ ID NO: 1864; antisense strand SEQ ID NO:1866) compoundtargeting APP (top) and a table showing the levels of AD-454972 compounddelivery in tissue at day 29 post IT administration in cyno monkeys of72 mg of AD-454972 targeting APP (bottom).

FIG. 6 is two graphs showing the results of CSF soluble APP alpha andbeta (top) and CSF amyloid beta species (bottom) collected 2-3 monthspost IT administration in cyno monkeys of 72 mg of AD-454972 targetingAPP.

FIG. 7A is two graphs showing the results of CSF collected at days 8,15, and 29 and analyzed for soluble APP alpha and beta(top) and amyloidbeta 38,40, and 42 (bottom), post IT administration in cyno monkeys of72 mg of AD-454842 targeting APP.

FIG. 7B is a table showing the levels of AD-454842 compound delivery intissue at day 29 post IT administration in cyno monkeys of 72 mg ofAD-454842 targeting APP.

FIG. 8A is two graphs showing the results of CSF collected at days 8,15, and 29 and analyzed for soluble APP alpha and beta (top) and amyloidbeta 38,40, and 42 (bottom), post IT administration in cyno monkeys of72 mg of AD-454843 targeting APP.

FIG. 8B is a graph showing the results of tissue mRNA knockdown at day29 post IT administration in cyno monkeys of 72 mg of AD-454843targeting APP.

FIG. 8C is a table showing the levels of AD-454843 compound delivery intissue at day 29 post IT administration in cyno monkeys of 72 mg ofAD-454843 targeting APP.

FIG. 9A is two graphs showing the results of CSF soluble APP alpha andbeta (top) and CSF amyloid beta species (bottom) collected 2-3 monthspost IT administration in cyno monkeys of 72 mg of AD-454843 targetingAPP.

FIG. 9B is a graph showing the results of tissue mRNA knockdown at day85 post IT administration in cyno monkeys of 72 mg of AD-454843targeting APP.

FIG. 10A is two graphs showing the results CSF collected at days 8, 15,and 29 and analyzed for soluble APP alpha and beta (top) and amyloidbeta 38,40, and 42 (bottom), post IT administration in cyno monkeys of72 mg of AD-454844 targeting APP.

FIG. 10B is a graph showing the results of tissue mRNA knockdown at day29 post IT administration in cyno monkeys of 72 mg of AD-454844targeting APP.

FIG. 10C is a scheme demonstrating the structure of the AD-454844 (sensestrand SEQ ID NO: 1880; antisense strand SEQ ID NO:1882) compoundtargeting APP (top) and a table showing the levels of AD-454844 compounddelivery in tissue at day 29 post IT administration in cyno monkeys of72 mg of AD-454844 targeting APP (bottom).

FIG. 11A is a table showing a high level of compound delivery in tissueat day 29 post IT administration in cyno monkeys of 72 mg siRNAtargeting APP.

FIG. 11B is a graph showing the results of tissue mRNA knockdown at day29 post IT administration in cyno monkeys of a high level (FIG. 11A) ofcompound delivery targeting APP.

FIG. 11C is two graphs showing the results of CSF collected at days 8,15, and 29 and analyzed for soluble APP alpha and beta(top) and amyloidbeta 38,40, and 42 (bottom), post IT administration in cyno monkeys of72 mg of of a high level of compound delivery (FIG. 11A) targeting APP.

FIG. 12A is two plots showing the average of 5 miRNA duplex studies. Toppanel is a box plot of the results of 5 compounds at day at day 29 postIT administration in cyno monkeys of 72 mg siRNA. Bottom panel is a boxplot of the amount of mRNA remaining in each tissue relative to acontrol 29 days post IT administration in cyno monkeys.

FIG. 12B is two two plots showing repeated miRNA duplex studies in whichCSF was collected at days 8, 15, and 29 and analyzed for soluble APPalpha and beta (top) and amyloid beta 38,40, and 42 (bottom), post ITadministration in cyno monkeys of 72 mg of siRNA compounds targetingAPP.

FIG. 13A is a graph demonstrating the percent APP mRNA remaining instriatum tissue 29 days post IT administration in cyno monkeys ofAD-454972 targeting APP.

FIG. 13B is a graph demonstrating the percent APP mRNA remaining instriatum tissue 29 days post IT administration in cyno monkeys ofAD-454973 targeting APP.

FIG. 13C is a graph demonstrating the percent APP mRNA remaining instriatum tissue 29 days post IT administration in cyno monkeys ofAD-454842 targeting APP.

FIG. 13D is a graph demonstrating the percent APP mRNA remaining instriatum tissue 29 days post IT administration in cyno monkeys ofAD-454843 targeting APP.

FIG. 13E is a graph demonstrating the percent APP mRNA remaining instriatum tissue 29 days post IT administration in cyno monkeys ofAD-454844 targeting APP.

FIG. 14A and FIG. 14B are schematic images of modified RNAi agentshaving AU-rich seeds that were screened for in vivo hsAPP knockdownactivity in mice. The agents include AD-392927 (sense strand SEQ IDNO:1141; antisense strand SEQ ID NO:1142), AD-506935 (sense strand SEQID NO:1916; antisense strand SEQ ID NO:1917), AD-507065 (sense strandSEQ ID NO:1918; antisense strand SEQ ID NO:1919), AD-507159 (sensestrand SEQ ID NO:1920; antisense strand SEQ ID NO:1921), AD-507538(sense strand SEQ ID NO:1922; antisense strand SEQ ID NO:1923),AD-507624 (sense strand SEQ ID NO:1924; antisense strand SEQ IDNO:1925), AD-507724 (sense strand SEQ ID NO:1926; antisense strand SEQID NO:1927), AD-507725 (sense strand SEQ ID NO:1928; antisense strandSEQ ID NO:1929), AD-507789 (sense strand SEQ ID NO:1930; antisensestrand SEQ ID NO:1931), AD-507874 (sense strand SEQ ID NO:1932;antisense strand SEQ ID NO:1933), AD-507928 (sense strand SEQ IDNO:1934; antisense strand SEQ ID NO:1935), and AD-507949 (sense strandSEQ ID NO:1936; antisense strand SEQ ID NO:1937).

FIG. 15 is a graph depicting % hs APP knockdown in the liver ofAAV8.HsAPP-CDS3TRNC mice treated with AU-rich seeds. PBS, Naïve, andAD-392927 (RLD592) controls are included in the graph.

FIG. 16A-16D are schematic images of modified lead RNAi agents that werescreened for in vivo hsAPP knockdown activity in AAV mice. The agentsinclude AD-886864 (sense strand SEQ ID NO:2598, antisense strand SEQ IDNO:2599), AD-886877 (sense strand SEQ ID NO:2624, antisense strand SEQID NO:2625), AD-886879 (sense strand SEQ ID NO:2628, antisense strandSEQ ID NO:2629), AD-886883 (sense strand SEQ ID NO:2636, antisensestrand SEQ ID NO:2637), AD-886884 (sense strand SEQ ID NO:2638,antisense strand SEQ ID NO:2639), AD-886889 (sense strand SEQ IDNO:2648, antisense strand SEQ ID NO:2649), AD-886899 (sense strand SEQID NO:2668, antisense strand SEQ ID NO:2669), AD-886900 (sense strandSEQ ID NO:2670, antisense strand SEQ ID NO:2671), AD-886907 (sensestrand SEQ ID NO:2684, antisense strand SEQ ID NO:2685), AD-886908(sense strand SEQ ID NO:2686, antisense strand SEQ ID NO:2687),AD-886919 (sense strand SEQ ID NO:2708, antisense strand SEQ IDNO:2709), AD-886930 (sense strand SEQ ID NO:2730, antisense strand SEQID NO:2731), AD-886931 (sense strand SEQ ID NO:2732, antisense strandSEQ ID NO:2733), AD-886823 (sense strand SEQ ID NO:2516, antisensestrand SEQ ID NO:2517), AD-886853 (sense strand SEQ ID NO:2576,antisense strand SEQ ID NO:2577), AD-886858 (sense strand SEQ IDNO:2586, antisense strand SEQ ID NO:2587), AD-886839 (sense strand SEQID NO:2546, antisense strand SEQ ID NO:2547), AD-886845 (sense strandSEQ ID NO:2560, antisense strand SEQ ID NO:2561), AD-886873 (sensestrand SEQ ID NO:2616, antisense strand SEQ ID NO:2617), AD-886906(sense strand SEQ ID NO:2682, antisense strand SEQ ID NO:2683),AD-886909 (sense strand SEQ ID NO:2688, antisense strand SEQ IDNO:2689), and AD-886928 (sense strand SEQ ID NO:2726, antisense strandSEQ ID NO:2727).

FIG. 17A and FIG. 17B are graphs depicting % hs APP knockdown in theliver of AAV8.HsAPP-CDS3TRNC mice treated with lead oligonucleotides.PBS and Naïve, controls are included in the graphs.

FIGS. 18A-18D are schematic images of modified lead RNAi agents thatwere screened for in vivo hsAPP knockdown activity in AAV mice and whichare grouped as families based on the AD-886864 parent (FIG. 18A),AD-886899 parent (FIG. 18B), AD-886919 parent (FIG. 18C), and AD-886823parent (FIG. 18D), respectively. The screened agents include AD-886864(sense strand SEQ ID NO:2598, antisense strand SEQ ID NO:2599),AD-886873 (sense strand SEQ ID NO:2616, antisense strand SEQ IDNO:2617), AD-886877 (sense strand SEQ ID NO:2624, antisense strand SEQID NO:2625), AD-886879 (sense strand SEQ ID NO:2628, antisense strandSEQ ID NO:2629), AD-886883 (sense strand SEQ ID NO:2636, antisensestrand SEQ ID NO:2637), AD-886884 (sense strand SEQ ID NO:2638,antisense strand SEQ ID NO:2639), AD-886889 (sense strand SEQ IDNO:2648, antisense strand SEQ ID NO:2649), AD-886899 (sense strand SEQID NO:2668, antisense strand SEQ ID NO:2669), AD-886900 (sense strandSEQ ID NO:2670, antisense strand SEQ ID NO:2671), AD-886906 (sensestrand SEQ ID NO:2682, antisense strand SEQ ID NO:2683), AD-886907(sense strand SEQ ID NO:2684, antisense strand SEQ ID NO:2685),AD-886908 (sense strand SEQ ID NO:2686, antisense strand SEQ IDNO:2687), AD-886909 (sense strand SEQ ID NO:2688, antisense strand SEQID NO:2689), AD-886919 (sense strand SEQ ID NO:2708, antisense strandSEQ ID NO:2709), AD-886928 (sense strand SEQ ID NO:2726, antisensestrand SEQ ID NO:2727), AD-886930 (sense strand SEQ ID NO:2730,antisense strand SEQ ID NO:2731), AD-886931 (sense strand SEQ IDNO:2732, antisense strand SEQ ID NO:2733), AD-886823 (sense strand SEQID NO:2516, antisense strand SEQ ID NO:2517), AD-886853 (sense strandSEQ ID NO:2576, antisense strand SEQ ID NO:2577), AD-886858 (sensestrand SEQ ID NO:2586, antisense strand SEQ ID NO:2587), AD-886839(sense strand SEQ ID NO:2546, antisense strand SEQ ID NO:2547), andAD-88845 (sense strand SEQ ID NO:2560; antisense strand SEQ ID NO:2561).

FIG. 19 is a scheme demonstrating the APP knock down non-human primate(NHP) screening study design of the AD-454844 4 month study in which asingle intrathecal (IT) injection of 60 mg of the compound of interestwas given to Cyno monkeys at the onset.

FIGS. 20A-20G 6 show data from in vivo screens of C16 siRNA conjugates,including the parent AD-454855, and 5 additional siRNA conjugatesderived from structure activity relationship studies of AD-454855.Graphs depict the percent soluble APP alpha and beta collected from theCSF on days 8, 15, and 19 post intrathecal administration of 60 mg ofeach compound. FIG. 20A is a graph of soluble APP alpha and beta 4months post dose of AD-454844 for two non-human primate subjects. FIG.20B is a graph depicting the percent soluble APP alpha and betacollected from the CSF at Days 8, 15, and 19 post dose of AD-454844.FIG. 20C is a graph depicting the percent soluble APP alpha and betacollected from the CSF at Days 8, 15, and 19 post dose of the 5′terminal C16 siRNA conjugate, AD-994379. FIG. 20D is a graph depictingthe percent soluble APP alpha and beta collected from the CSF at Days 8,15, and 19 post dose of AD-961583. FIG. 20E is a graph depicting thepercent soluble APP alpha and beta collected from the CSF at Days 8, 15,and 19 post dose of AD-961584. FIG. 20F is a graph depicting the percentsoluble APP alpha and beta collected from the CSF at Days 8, 15, and 19post dose of AD-961585. FIG. 20G is a graph depicting the percentsoluble APP alpha and beta collected from the CSF at Days 8, 15, and 19post dose of AD-961586.

FIGS. 21A and 21B are schematic images of C16 modified lead RNAi agentsthat were screened for in vivo APP knockdown activity in non-humanprimates. FIG. 21A is a schematic of the parent internal C16 RNAi agentAD-454844 (sense strand SEQ ID NO:1880; antisense strand SEQ ID NO:1882)and the 5′ terminal C16 siRNA agent AD-994379 (sense strand SEQ IDNO:2893; antisense strand SEQ ID NO:2894). FIG. 21B is a schematic ofRNAi agents AD-961583 (sense strand SEQ ID NO:2895; antisense strand SEQID NO:2896), AD-961584 (sense strand SEQ ID NO:2897; antisense strandSEQ ID NO:2898), AD-961585 (sense strand SEQ ID NO:2899; antisensestrand SEQ ID NO:2900), and AD-961586(sense strand SEQ ID NO:2901;antisense strand SEQ ID NO:2902).

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides RNAi compositions, which effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of an amyloid precursor protein (APP) gene. The APP gene maybe within a cell, e.g., a cell within a subject, such as a human. Thepresent disclosure also provides methods of using the RNAi compositionsof the disclosure for inhibiting the expression of an APP gene and/orfor treating a subject having a disorder that would benefit frominhibiting or reducing the expression of an APP gene, e.g., anAPP-associated disease, for example, cerebral amyloid angiopathy (CAA)or Alzheimer's disease (AD), e.g., early onset familial Alzheimerdisease (EOFAD).

The RNAi agents of the disclosure include an RNA strand (the antisensestrand) having a region which is about 30 nucleotides or less in length,e.g., 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22,15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26,18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27,19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28,20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28,21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides in length, whichregion is substantially complementary to at least part of an mRNAtranscript of an APP gene.

In certain embodiments, the RNAi agents of the disclosure include an RNAstrand (the antisense strand) which can include longer lengths, forexample up to 66 nucleotides, e.g., 36-66, 26-36, 25-36, 31-60, 22-43,27-53 nucleotides in length with a region of at least 19 contiguousnucleotides that is substantially complementary to at least a part of anmRNA transcript of an APP gene. These RNAi agents with the longer lengthantisense strands preferably include a second RNA strand (the sensestrand) of 20-60 nucleotides in length wherein the sense and antisensestrands form a duplex of 18-30 contiguous nucleotides.

The use of these RNAi agents enables the targeted degradation of mRNAsof an APP gene in mammals. Very low dosages of APP RNAi agents, inparticular, can specifically and efficiently mediate RNA interference(RNAi), resulting in significant inhibition of expression of an APPgene. Using cell-based assays, the present inventors have demonstratedthat RNAi agents targeting APP can mediate RNAi, resulting insignificant inhibition of expression of an APP gene. Thus, methods andcompositions including these RNAi agents are useful for treating asubject who would benefit by a reduction in the levels and/or activityof an APP protein, such as a subject having an APP-associated disease,for example, CAA or AD, including, e.g., EOFAD.

The following detailed description discloses how to make and usecompositions containing RNAi agents to inhibit the expression of an APPgene, as well as compositions and methods for treating subjects havingdiseases and disorders that would benefit from inhibition and/orreduction of the expression of this gene.

I. Definitions

In order that the present disclosure may be more readily understood,certain terms are first defined. In addition, it should be noted thatwhenever a value or range of values of a parameter are recited, it isintended that values and ranges intermediate to the recited values arealso intended to be part of this disclosure.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element, e.g., a plurality of elements.

The term “including” is used herein to mean, and is used interchangeablywith, the phrase “including but not limited to”. The term “or” is usedherein to mean, and is used interchangeably with, the term “and/or,”unless context clearly indicates otherwise.

The term “about” is used herein to mean within the typical ranges oftolerances in the art. For example, “about” can be understood as about 2standard deviations from the mean. In certain embodiments, aboutmeans±10%. In certain embodiments, about means±5%. When about is presentbefore a series of numbers or a range, it is understood that “about” canmodify each of the numbers in the series or range.

The term “at least” prior to a number or series of numbers is understoodto include the number adjacent to the term “at least”, and allsubsequent numbers or integers that could logically be included, asclear from context. For example, the number of nucleotides in a nucleicacid molecule must be an integer. For example, “at least 18 nucleotidesof a 21 nucleotide nucleic acid molecule” means that 18, 19, 20, or 21nucleotides have the indicated property. When at least is present beforea series of numbers or a range, it is understood that “at least” canmodify each of the numbers in the series or range.

As used herein, “no more than” or “less than” is understood as the valueadjacent to the phrase and logical lower values or intergers, as logicalfrom context, to zero. For example, a duplex with an overhang of “nomore than 2 nucleotides” has a 2, 1, or 0 nucleotide overhang. When “nomore than” is present before a series of numbers or a range, it isunderstood that “no more than” can modify each of the numbers in theseries or range.

The term “APP” amyloid precursor protein (APP), also known as amyloidbeta precursor protein, Alzheimer disease amyloid protein and cerebralvascular amyloid peptide, among other names, having an amino acidsequence from any vertebrate or mammalian source, including, but notlimited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine,primate, monkey, and guinea pig, unless specified otherwise. The termalso refers to fragments and variants of native APP that maintain atleast one in vivo or in vitro activity of a native APP (including, e.g.,the beta-amyloid peptide(1-40), beta-amyloid peptide(1-38) andbeta-amyloid peptide(1-42) forms of Aβ peptide, among others), includingvariants of APP fragments that maintain one or more activities of an APPfragment that are neurotoxic in character (e.g., variant forms of Aβ42peptide that maintain neurotoxic character are expressly contemplated).The term encompasses full-length unprocessed precursor forms of APP aswell as mature forms resulting from post-translational cleavage of thesignal peptide. The term also encompasses peptides that derive from APPvia further cleavage, including, e.g., Aβ peptides. The nucleotide andamino acid sequence of a human APP can be found at, for example, GenBankAccession No. GI: 228008405 (NM_201414; SEQ ID NO: 1). The nucleotideand amino acid sequence of a human APP may also be found at, forexample, GenBank Accession No. GI: 228008403 (NM_000484.3; SEQ ID NO:2); GenBank Accession No. GI: 228008404 (NM_201413.2; SEQ ID NO: 3);GenBank Accession No. GI: 324021746 (NM_001136016.3; SEQ ID NO: 4);GenBank Accession No. GI: 228008402 (NM_001136129.2; SEQ ID NO: 5);GenBank Accession No. GI: 228008401 (NM_001136130.2; SEQ ID NO: 6);GenBank Accession No. GI: 324021747 (NM_001136131.2; SEQ ID NO: 7);GenBank Accession No. GI: 324021737 (NM_001204301.1; SEQ ID NO: 8);GenBank Accession No. GI: 324021735 (NM_001204302.1; SEQ ID NO: 9); andGenBank Accession No. GI: 324021739 (NM_001204303.1; SEQ ID NO: 10); andGenBank Accession No. GI: 1370481385 (XM_024452075.1; SEQ ID NO: 11).

The nucleotide and amino acid sequence of a Cynomolgus monkey APP can befound at, for example, GenBank Accession No. GI: 982237868(XM_005548883.2; SEQ ID NO: 12). The nucleotide and amino acid sequenceof a mouse APP can be found at, for example, GenBank Accession No. GI:311893400 (NM_001198823; SEQ ID NO: 13). The nucleotide and amino acidsequence of a rat APP can be found at, for example, GenBank AccessionNo. GI: 402692725 (NM_019288.2; SEQ ID NO: 14). Additional examples ofAPP sequences are readily available using publicly available databases,e.g., GenBank, UniProt, and OMIM.

The term“APP” as used herein also refers to a particular polypeptideexpressed in a cell by naturally occurring DNA sequence variations ofthe APP gene, such as a single nucleotide polymorphism in the APP gene.Numerous SNPs within the APP gene have been identified and may be foundat, for example, NCBI dbSNP (see, e.g., www.ncbi.nlm.nih.gov/snp).Non-limiting examples of SNPs within the APP gene may be found at, NCBIdbSNP Accession Nos. rs193922916, rs145564988, rs193922916, rs214484,rs281865161, rs364048, rs466433, rs466448, rs532876832, rs63749810,rs63749964, rs63750064, rs63750066, rs63750151, rs63750264, rs63750363,rs63750399, rs63750445, rs63750579, rs63750643, rs63750671, rs63750734,rs63750847, rs63750851, rs63750868, rs63750921, rs63750973, rs63751039,rs63751122 and rs63751263. Certain exemplary rare APP variants that havebeen previously described to play a role in development of EOFAD wereidentified in Hooli et al. (Neurology 78: 1250-57). In addition, various“non-classical” APP variants that harbor an intraexonic junction withinsequenced cDNA have recently been identified as associated with theoccurrence of somatic gene recombination in the brains of AD patients(PCT/US2018/030520, which is incorporated herein by reference in itsentirety). Examples of such “non-classical” APP variants includecAPP-R3/16 (SEQ ID NO: 1865), cAPP-R3/16-2 (SEQ ID NO: 1866), cAPP-R2/18(SEQ ID NO: 1867), cAPP-R6/18 (SEQ ID NO: 1868), cAPP-R3/14 (SEQ ID NO:1869), cAPP-R3/17 (SEQ ID NO: 1870), cAPP-R1/11 (SEQ ID NO: 1871),cAPP-R1/13 (SEQ ID NO: 1872), cAPP-R1/11-2 (SEQ ID NO: 1873), cAPP-R1/14(SEQ ID NO: 1874), cAPP-R2/17 (SEQ ID NO: 1875), cAPP-R2/16 (SEQ ID NO:1876), cAPP-R6/17 (SEQ ID NO: 1877), cAPP-R2/14 (SEQ ID NO: 1878),cAPP-R14/17-d8 (SEQ ID NO: 1879) and cAPP-D2/18-3 (SEQ ID NO: 1880). Itis expressly contemplated that RNAi agents of the instant disclosure canbe used to target “non-classical” APP variants and/or that RNAi agentsoptionally specific for such “non-classical” APP variants can bedesigned and used, optionally in combination with other RNAi agents ofthe instant disclosure, including those that target native forms of APP.Such “non-classical” APP variants were described as notably absent froman assayed HIV patient population, with prevalence of AD in the HIVpatient population significantly diminished as compared to expectedlevels, which indicated that reverse transcriptase inhibitors and/orother anti-retroviral therapies commonly used to treat HIV patientslikely also exerted a therapeutic/preventative role against AD. It istherefore expressly contemplated that the RNAi agents of the instantdisclosure can optionally be employed in combination with reversetranscriptase inhibitors and/or other anti-retroviral therapies, fortherapeutic and/or preventative purposes.

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an APP gene, including mRNA that is a product of RNA processing of aprimary transcription product. In one embodiment, the target portion ofthe sequence will be at least long enough to serve as a substrate forRNAi-directed cleavage at or near that portion of the nucleotidesequence of an mRNA molecule formed during the transcription of an APPgene.

The target sequence may be from about 9-36 nucleotides in length, e.g.,about 15-30 nucleotides in length. For example, the target sequence canbe from about 15-30 nucleotides, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22nucleotides in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of thedisclosure.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacement moiety(see, e.g., Table 1). The skilled person is well aware that guanine,cytosine, adenine, thymidine, and uracil can be replaced by othermoieties without substantially altering the base pairing properties ofan oligonucleotide comprising a nucleotide bearing such replacementmoiety. For example, without limitation, a nucleotide comprising inosineas its base can base pair with nucleotides containing adenine, cytosine,or uracil. Hence, nucleotides containing uracil, guanine, or adenine canbe replaced in the nucleotide sequences of dsRNA featured in thedisclosure by a nucleotide containing, for example, inosine. In anotherexample, adenine and cytosine anywhere in the oligonucleotide can bereplaced with guanine and uracil, respectively to form G-U Wobble basepairing with the target mRNA. Sequences containing such replacementmoieties are suitable for the compositions and methods featured in thedisclosure.

The terms “iRNA”, “RNAi agent,” “iRNA agent,” “RNA interference agent”as used interchangeably herein, refer to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.RNA interference (RNAi) is a process that directs the sequence-specificdegradation of mRNA. RNAi modulates, e.g., inhibits, the expression ofAPP in a cell, e.g., a cell within a subject, such as a mammaliansubject.

In one embodiment, an RNAi agent of the disclosure includes a singlestranded RNAi that interacts with a target RNA sequence, e.g., an APPtarget mRNA sequence, to direct the cleavage of the target RNA. Withoutwishing to be bound by theory it is believed that long double strandedRNA introduced into cells is broken down into double-stranded shortinterfering RNAs (siRNAs) comprising a sense strand and an antisensestrand by a Type III endonuclease known as Dicer (Sharp et al. (2001)Genes Dev. 15:485). Dicer, a ribonuclease-III-like enzyme, processesthese dsRNA into 19-23 base pair short interfering RNAs withcharacteristic two base 3′ overhangs (Bernstein, et al., (2001) Nature409:363). These siRNAs are then incorporated into an RNA-inducedsilencing complex (RISC) where one or more helicases unwind the siRNAduplex, enabling the complementary antisense strand to guide targetrecognition (Nykanen, et al., (2001) Cell 107:309). Upon binding to theappropriate target mRNA, one or more endonucleases within the RISCcleave the target to induce silencing (Elbashir, et al., (2001) GenesDev. 15:188). Thus, in one aspect the disclosure relates to a singlestranded RNA (ssRNA) (the antisense strand of a siRNA duplex) generatedwithin a cell and which promotes the formation of a RISC complex toeffect silencing of the target gene, i.e., an APP gene. Accordingly, theterm “siRNA” is also used herein to refer to an RNAi as described above.

In another embodiment, the RNAi agent may be a single-stranded RNA thatis introduced into a cell or organism to inhibit a target mRNA.Single-stranded RNAi agents bind to the RISC endonuclease, Argonaute 2,which then cleaves the target mRNA. The single-stranded siRNAs aregenerally 15-30 nucleotides and are chemically modified. The design andtesting of single-stranded RNAs are described in U.S. Pat. No. 8,101,348and in Lima et al., (2012) Cell 150:883-894, the entire contents of eachof which are hereby incorporated herein by reference. Any of theantisense nucleotide sequences described herein may be used as asingle-stranded siRNA as described herein or as chemically modified bythe methods described in Lima et al., (2012) Cell 150:883-894.

In another embodiment, a “RNAi agent” for use in the compositions andmethods of the disclosure is a double stranded RNA and is referred toherein as a “double stranded RNAi agent,” “double stranded RNA (dsRNA)molecule,” “dsRNA agent,” or “dsRNA”. The term “dsRNA” refers to acomplex of ribonucleic acid molecules, having a duplex structurecomprising two anti-parallel and substantially complementary nucleicacid strands, referred to as having “sense” and “antisense” orientationswith respect to a target RNA, i.e., an APP gene. In some embodiments ofthe disclosure, a double stranded RNA (dsRNA) triggers the degradationof a target RNA, e.g., an mRNA, through a post-transcriptionalgene-silencing mechanism referred to herein as RNA interference or RNAi.

In general, a number of nucleotides of each strand of a dsRNA moleculeare ribonucleotides, but as described in detail herein, each or bothstrands can also include one or more non-ribonucleotides, e.g., adeoxyribonucleotide and/or a modified nucleotide. In addition, as usedin this specification, an “RNAi agent” may include ribonucleotides withchemical modifications; an RNAi agent may include substantialmodifications at multiple nucleotides. As used herein, the term“modified nucleotide” refers to a nucleotide having, independently, amodified sugar moiety, a modified internucleotide linkage, and/or amodified nucleobase. Thus, the term modified nucleotide encompassessubstitutions, additions or removal of, e.g., a functional group oratom, to internucleoside linkages, sugar moieties, or nucleobases. Themodifications suitable for use in the agents of the disclosure includeall types of modifications disclosed herein or known in the art. Anysuch modifications, as used in a siRNA type molecule, are encompassed by“RNAi agent” for the purposes of this specification and claims.

In certain embodiments of the instant disclosure, inclusion of adeoxy-nucleotide—which is acknowledged as a naturally occurring form ofnucleotide—if present within a RNAi agent can be considered toconstitute a modified nucleotide.

The duplex region may be of any length that permits specific degradationof a desired target RNA through a RISC pathway, and may range from about9 to 36 base pairs in length, e.g., about 15-30 base pairs in length,for example, about 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, or 36 base pairsin length, such as about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25,15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29,18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30,19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20,20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21,21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 basepairs in length. Ranges and lengths intermediate to the above recitedranges and lengths are also contemplated to be part of the disclosure.

The two strands forming the duplex structure may be different portionsof one larger RNA molecule, or they may be separate RNA molecules. Wherethe two strands are part of one larger molecule, and therefore areconnected by an uninterrupted chain of nucleotides between the 3′-end ofone strand and the 5′-end of the respective other strand forming theduplex structure, the connecting RNA chain is referred to as a “hairpinloop.” A hairpin loop can comprise at least one unpaired nucleotide. Insome embodiments, the hairpin loop can comprise at least 2, at least 3,at least 4, at least 5, at least 6, at least 7, at least 8, at least 9,at least 10, at least 20, at least 23 or more unpaired nucleotides. Insome embodiments, the hairpin loop can be 10 or fewer nucleotides. Insome embodiments, the hairpin loop can be 8 or fewer unpairednucleotides. In some embodiments, the hairpin loop can be 4-10 unpairednucleotides. In some embodiments, the hairpin loop can be 4-8nucleotides.

Where the two substantially complementary strands of a dsRNA arecomprised by separate RNA molecules, those molecules need not, but canbe covalently connected. In certain embodiments where the two strandsare connected covalently by means other than an uninterrupted chain ofnucleotides between the 3′-end of one strand and the 5′-end of therespective other strand forming the duplex structure, the connectingstructure is referred to as a “linker” (though it is noted that certainother structures defined elsewhere herein can also be referred to as a“linker”). The RNA strands may have the same or a different number ofnucleotides. The maximum number of base pairs is the number ofnucleotides in the shortest strand of the dsRNA minus any overhangs thatare present in the duplex. In addition to the duplex structure, an RNAimay comprise one or more nucleotide overhangs. In one embodiment of theRNAi agent, at least one strand comprises a 3′ overhang of at least 1nucleotide. In another embodiment, at least one strand comprises a 3′overhang of at least 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11,12, 13, 14, or 15 nucleotides. In other embodiments, at least one strandof the RNAi agent comprises a 5′ overhang of at least 1 nucleotide. Incertain embodiments, at least one strand comprises a 5′ overhang of atleast 2 nucleotides, e.g., 2, 3, 4, 5, 6, 7, 9, 10, 11, 12, 13, 14, or15 nucleotides. In still other embodiments, both the 3′ and the 5′ endof one strand of the RNAi agent comprise an overhang of at least 1nucleotide.

In one embodiment, an RNAi agent of the disclosure is a dsRNA, eachstrand of which comprises 19-23 nucleotides, that interacts with atarget RNA sequence, e.g., an APP target mRNA sequence, to direct thecleavage of the target RNA. Without wishing to be bound by theory, longdouble stranded RNA introduced into cells is broken down into siRNA by aType III endonuclease known as Dicer (Sharp et al. (2001) Genes Dev.15:485). Dicer, a ribonuclease-III-like enzyme, processes the dsRNA into19-23 base pair short interfering RNAs with characteristic two base 3′overhangs (Bernstein, et al., (2001) Nature 409:363). The siRNAs arethen incorporated into an RNA-induced silencing complex (RISC) where oneor more helicases unwind the siRNA duplex, enabling the complementaryantisense strand to guide target recognition (Nykanen, et al., (2001)Cell 107:309). Upon binding to the appropriate target mRNA, one or moreendonucleases within the RISC cleave the target to induce silencing(Elbashir, et al., (2001) Genes Dev. 15:188).

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of a RNAiagent, e.g., a dsRNA. For example, when a 3′-end of one strand of adsRNA extends beyond the 5′-end of the other strand, or vice versa,there is a nucleotide overhang. A dsRNA can comprise an overhang of atleast one nucleotide; alternatively the overhang can comprise at leasttwo nucleotides, at least three nucleotides, at least four nucleotides,at least five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) can be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′-end, 3′-end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide,overhang at the 3′-end and/or the 5′-end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide, e.g., a 1, 2, 3, 4, 5, 6, 7, 8,9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end. Inanother embodiment, one or more of the nucleotides in the overhang isreplaced with a nucleoside thiophosphate.

In certain embodiments, the antisense strand of a dsRNA has a 1-10nucleotide, e.g., 0-3, 1-3, 2-4, 2-5, 4-10, 5-10, e.g., a 1, 2, 3, 4, 5,6, 7, 8, 9, or 10 nucleotide, overhang at the 3′-end and/or the 5′-end.In one embodiment, the sense strand of a dsRNA has a 1-10 nucleotide,e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide, overhang at the3′-end and/or the 5′-end. In another embodiment, one or more of thenucleotides in the overhang is replaced with a nucleoside thiophosphate.

In certain embodiments, the overhang on the sense strand or theantisense strand, or both, can include extended lengths longer than 10nucleotides, e.g., 1-30 nucleotides, 2-30 nucleotides, 10-30nucleotides, or 10-15 nucleotides in length. In certain embodiments, anextended overhang is on the sense strand of the duplex. In certainembodiments, an extended overhang is present on the 3′end of the sensestrand of the duplex. In certain embodiments, an extended overhang ispresent on the 5′end of the sense strand of the duplex. In certainembodiments, an extended overhang is on the antisense strand of theduplex. In certain embodiments, an extended overhang is present on the3′end of the antisense strand of the duplex. In certain embodiments, anextended overhang is present on the 5′end of the antisense strand of theduplex. In certain embodiments, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate. In certainembodiments, the overhang includes a self-complementary portion suchthat the overhang is capable of forming a hairpin structure that isstable under physiological conditions.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will be doublestranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aRNAi agent, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence, e.g., an APP mRNA.

As used herein, the term “region of complementarity” refers to theregion on the antisense strand that is substantially complementary to asequence, for example a target sequence, e.g., an APP nucleotidesequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches can be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′- and/or 3′-terminus of the RNAi agent.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of a RNAi agent that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “cleavage region” refers to a region that islocated immediately adjacent to the cleavage site. The cleavage site isthe site on the target at which cleavage occurs. In some embodiments,the cleavage region comprises three bases on either end of, andimmediately adjacent to, the cleavage site. In some embodiments, thecleavage region comprises two bases on either end of, and immediatelyadjacent to, the cleavage site. In some embodiments, the cleavage sitespecifically occurs at the site bound by nucleotides 10 and 11 of theantisense strand, and the cleavage region comprises nucleotides 11, 12and 13.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions can include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing (see, e.g.,“Molecular Cloning: A Laboratory Manual, Sambrook, et al. (1989) ColdSpring Harbor Laboratory Press). Other conditions, such asphysiologically relevant conditions as can be encountered inside anorganism, can apply. The skilled person will be able to determine theset of conditions most appropriate for a test of complementarity of twosequences in accordance with the ultimate application of the hybridizednucleotides.

Complementary sequences within a RNAi agent, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they can form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,can yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, can also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in so far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs include, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein can be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of a RNAi agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding APP). For example, a polynucleotide iscomplementary to at least a part of an APP mRNA if the sequence issubstantially complementary to a non-interrupted portion of an mRNAencoding APP.

Accordingly, in some embodiments, the antisense strand polynucleotidesdisclosed herein are fully complementary to the target APP sequence. Inother embodiments, the antisense strand polynucleotides disclosed hereinare substantially complementary to the target APP sequence and comprisea contiguous nucleotide sequence which is at least about 80%complementary over its entire length to the equivalent region of thenucleotide sequence of SEQ ID NOs: 1-14, or a fragment of SEQ ID NOs:1-14, such as about 85%, about 86%, about 87%, about 88%, about 89%,about 90%, about % 91%, about 92%, about 93%, about 94%, about 95%,about 96%, about 97%, about 98%, or about 99% complementary.

In other embodiments, the antisense polynucleotides disclosed herein aresubstantially complementary to the target APP sequence and comprise acontiguous nucleotide sequence which is at least about 80% complementaryover its entire length to any one of the sense strand nucleotidesequences in any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B,or 26, or a fragment of any one of the sense strand nucleotide sequencesin any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, or 26,such as about 85%, about 86%, about 87%, about 88%, about 89%, about90%, about % 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, or about 99% complementary.

In one embodiment, an RNAi agent of the disclosure includes a sensestrand that is substantially complementary to an antisensepolynucleotide which, in turn, is the same as a target APP sequence, andwherein the sense strand polynucleotide comprises a contiguousnucleotide sequence which is at least about 80% complementary over itsentire length to the equivalent region of the nucleotide sequence of SEQID NOs: 15-28, or a fragment of any one of SEQ ID NOs: 15-28, such asabout 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about% 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%,about 98%, or about 99% complementary.

In one embodiment, at least partial suppression of the expression of anAPP gene, is assessed by a reduction of the amount of APP mRNA which canbe isolated from or detected in a first cell or group of cells in whichan APP gene is transcribed and which has or have been treated such thatthe expression of an APP gene is inhibited, as compared to a second cellor group of cells substantially identical to the first cell or group ofcells but which has or have not been so treated (control cells). Thedegree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

The phrase “contacting a cell with an RNAi agent,” such as a dsRNA, asused herein, includes contacting a cell by any possible means.Contacting a cell with an RNAi agent includes contacting a cell in vitrowith the RNAi agent or contacting a cell in vivo with the RNAi agent.The contacting may be done directly or indirectly. Thus, for example,the RNAi agent may be put into physical contact with the cell by theindividual performing the method, or alternatively, the RNAi agent maybe put into a situation that will permit or cause it to subsequentlycome into contact with the cell.

Contacting a cell in vitro may be done, for example, by incubating thecell with the RNAi agent. Contacting a cell in vivo may be done, forexample, by injecting the RNAi agent into or near the tissue where thecell is located, or by injecting the RNAi agent into another area, e.g.,the central nervous system (CNS), optionally via intrathecal,intravitreal or other injection, or to the bloodstream or thesubcutaneous space, such that the agent will subsequently reach thetissue where the cell to be contacted is located. For example, the RNAiagent may contain and/or be coupled to a ligand, e.g., a lipophilicmoiety or moieties as described below and further detailed, e.g., inU.S. Application Nos. 62/668,072, 62/738,747 and/or 62/773,082, thatdirects and/or otherwise stabilizes the RNAi agent at a site ofinterest, e.g., the CNS. Combinations of in vitro and in vivo methods ofcontacting are also possible. For example, a cell may also be contactedin vitro with an RNAi agent and subsequently transplanted into asubject.

In one embodiment, contacting a cell with a RNAi agent includes“introducing” or “delivering the RNAi agent into the cell” byfacilitating or effecting uptake or absorption into the cell. Absorptionor uptake of a RNAi agent can occur through unaided diffusive or activecellular processes, or by auxiliary agents or devices. Introducing aRNAi agent into a cell may be in vitro and/or in vivo. For example, forin vivo introduction, a RNAi agent can be injected into a tissue site oradministered systemically. In vitro introduction into a cell includesmethods known in the art such as electroporation and lipofection.Further approaches are described herein below and/or are known in theart.

The term “lipophile” or “lipophilic moiety” broadly refers to anycompound or chemical moiety having an affinity for lipids. One way tocharacterize the lipophilicity of the lipophilic moiety is by theoctanol-water partition coefficient, log K_(ow), where K_(ow) is theratio of a chemical's concentration in the octanol-phase to itsconcentration in the aqueous phase of a two-phase system at equilibrium.The octanol-water partition coefficient is a laboratory-measuredproperty of a substance. However, it may also be predicted by usingcoefficients attributed to the structural components of a chemical whichare calculated using first-principle or empirical methods (see, forexample, Tetko et al., J. Chem. Inf. Comput. Sci. 41:1407-21 (2001),which is incorporated herein by reference in its entirety). It providesa thermodynamic measure of the tendency of the substance to prefer anon-aqueous or oily milieu rather than water (i.e. itshydrophilic/lipophilic balance). In principle, a chemical substance islipophilic in character when its log K_(ow) exceeds 0. Typically, thelipophilic moiety possesses a log K_(ow) exceeding 1, exceeding 1.5,exceeding 2, exceeding 3, exceeding 4, exceeding 5, or exceeding 10. Forinstance, the log K_(ow) of 6-amino hexanol, for instance, is predictedto be approximately 0.7. Using the same method, the log K_(ow) ofcholesteryl N-(hexan-6-ol) carbamate is predicted to be 10.7.

The lipophilicity of a molecule can change with respect to thefunctional group it carries. For instance, adding a hydroxyl group oramine group to the end of a lipophilic moiety can increase or decreasethe partition coefficient (e.g., log K_(ow)) value of the lipophilicmoiety.

Alternatively, the hydrophobicity of the double-stranded RNAi agent,conjugated to one or more lipophilic moieties, can be measured by itsprotein binding characteristics. For instance, in certain embodiments,the unbound fraction in the plasma protein binding assay of thedouble-stranded RNAi agent could be determined to positively correlateto the relative hydrophobicity of the double-stranded RNAi agent, whichcould then positively correlate to the silencing activity of thedouble-stranded RNAi agent.

In one embodiment, the plasma protein binding assay determined is anelectrophoretic mobility shift assay (EMSA) using human serum albuminprotein. An exemplary protocol of this binding assay is illustrated indetail in, e.g., U.S. Application Nos. 62/668,072, 62/738,747 and/or62/773,082. The hydrophobicity of the double-stranded RNAi agent,measured by fraction of unbound siRNA in the binding assay, exceeds0.15, exceeds 0.2, exceeds 0.25, exceeds 0.3, exceeds 0.35, exceeds 0.4,exceeds 0.45, or exceeds 0.5 for an enhanced in vivo delivery of siRNA.

Accordingly, conjugating the lipophilic moieties to the internalposition(s) of the double-stranded RNAi agent provides optimalhydrophobicity for the enhanced in vivo delivery of siRNA.

The term “lipid nanoparticle” or “LNP” is a vesicle comprising a lipidlayer encapsulating a pharmaceutically active molecule, such as anucleic acid molecule, e.g., a rNAi agent or a plasmid from which a RNAiagent is transcribed. LNPs are described in, for example, U.S. Pat. Nos.6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents ofwhich are hereby incorporated herein by reference.

As used herein, a “subject” is an animal, such as a mammal, including aprimate (such as a human, a non-human primate, e.g., a monkey, and achimpanzee), a non-primate (such as a cow, a pig, a camel, a llama, ahorse, a goat, a rabbit, a sheep, a hamster, a guinea pig, a cat, a dog,a rat, a mouse, a horse, and a whale), or a bird (e.g., a duck or agoose). In an embodiment, the subject is a human, such as a human beingtreated or assessed for a disease, disorder or condition that wouldbenefit from reduction in APP expression; a human at risk for a disease,disorder or condition that would benefit from reduction in APPexpression; a human having a disease, disorder or condition that wouldbenefit from reduction in APP expression; and/or human being treated fora disease, disorder or condition that would benefit from reduction inAPP expression as described herein.

As used herein, the terms “treating” or “treatment” refer to abeneficial or desired result including, but not limited to, alleviationor amelioration of one or more symptoms associated with APP geneexpression and/or APP protein production, e.g., APP-associated diseasesor disorders such as AD, CAA (e.g., hereditary CAA) and EOFAD, amongothers. “Treatment” can also mean prolonging survival as compared toexpected survival in the absence of treatment.

The term “lower” in the context of the level of APP in a subject or adisease marker or symptom refers to a statistically significant decreasein such level. The decrease can be, for example, at least 10%, at least15%, at least 20%, at least 25%, at least 30%, at least 35%, at least40%, at least 45%, at least 50%, at least 55%, at least 60%, at least65%, at least 70%, at least 75%, at least 80%, at least 85%, at least90%, at least 95%, or more. In certain embodiments, a decrease is atleast 20%. “Lower” in the context of the level of APP in a subject ispreferably down to a level accepted as within the range of normal for anindividual without such disorder.

As used herein, “prevention” or “preventing,” when used in reference toa disease, disorder or condition thereof, that would benefit from areduction in expression of an APP gene and/or production of APP protein,refers to a reduction in the likelihood that a subject will develop asymptom associated with such a disease, disorder, or condition, e.g., asymptom of APP gene expression, such as the presence of various forms ofAP (e.g., A1338, Aβ40 and/or Aβ42, etc.), amyloid plaques and/orcerebral amyloid angiopathy (CAA) or Alzheimer's disease (AD),including, e.g., early onset familial Alzheimer disease (EOFAD). Thefailure to develop a disease, disorder or condition, or the reduction inthe development of a symptom associated with such a disease, disorder orcondition (e.g., by at least about 10% on a clinically accepted scalefor that disease or disorder), or the exhibition of delayed symptomsdelayed (e.g., by days, weeks, months or years) is considered effectiveprevention.

As used herein, the term “APP-associated disease,” is a disease ordisorder that is caused by, or associated with APP gene expression orAPP protein production. The term “APP-associated disease” includes adisease, disorder or condition that would benefit from a decrease in APPgene expression, replication, or protein activity. Non-limiting examplesof APP-associated diseases include, for example, cerebral amyloidangiopathy (CAA) and Alzheimer's disease (AD), including, e.g., earlyonset familial Alzheimer disease (EOFAD).

“Therapeutically effective amount,” as used herein, is intended toinclude the amount of an RNAi agent that, when administered to a subjecthaving an APP-associated disorder, is sufficient to effect treatment ofthe disease (e.g., by diminishing, ameliorating or maintaining theexisting disease or one or more symptoms of disease). The“therapeutically effective amount” may vary depending on the RNAi agent,how the agent is administered, the disease and its severity and thehistory, age, weight, family history, genetic makeup, the types ofpreceding or concomitant treatments, if any, and other individualcharacteristics of the subject to be treated.

“Prophylactically effective amount,” as used herein, is intended toinclude the amount of a RNAi agent that, when administered to a subjecthaving an APP-associated disorder, is sufficient to prevent orameliorate the disease or one or more symptoms of the disease.Ameliorating the disease includes slowing the course of the disease orreducing the severity of later-developing disease. The “prophylacticallyeffective amount” may vary depending on the RNAi agent, how the agent isadministered, the degree of risk of disease, and the history, age,weight, family history, genetic makeup, the types of preceding orconcomitant treatments, if any, and other individual characteristics ofthe patient to be treated.

A “therapeutically-effective amount” or “prophylacticaly effectiveamount” also includes an amount of a RNAi agent that produces somedesired local or systemic effect at a reasonable benefit/risk ratioapplicable to any treatment. A RNAi agent employed in the methods of thepresent disclosure may be administered in a sufficient amount to producea reasonable benefit/risk ratio applicable to such treatment.

The phrase “pharmaceutically acceptable” is employed herein to refer tothose compounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human subjects and animal subjects without excessivetoxicity, irritation, allergic response, or other problem orcomplication, commensurate with a reasonable benefit/risk ratio.

The phrase “pharmaceutically-acceptable carrier” as used herein means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the subject being treated. Some examples ofmaterials which can serve as pharmaceutically-acceptable carriersinclude: (1) sugars, such as lactose, glucose and sucrose; (2) starches,such as corn starch and potato starch; (3) cellulose, and itsderivatives, such as sodium carboxymethyl cellulose, ethyl cellulose andcellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7)lubricating agents, such as magnesium state, sodium lauryl sulfate andtalc; (8) excipients, such as cocoa butter and suppository waxes; (9)oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; (10) glycols, such as propyleneglycol; (11) polyols, such as glycerin, sorbitol, mannitol andpolyethylene glycol; (12) esters, such as ethyl oleate and ethyllaurate; (13) agar; (14) buffering agents, such as magnesium hydroxideand aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17)isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pHbuffered solutions; (21) polyesters, polycarbonates and/orpolyanhydrides; (22) bulking agents, such as polypeptides and aminoacids (23) serum component, such as serum albumin, HDL and LDL; and (22)other non-toxic compatible substances employed in pharmaceuticalformulations.

The term “sample,” as used herein, includes a collection of similarfluids, cells, or tissues isolated from a subject, as well as fluids,cells, or tissues present within a subject. Examples of biologicalfluids include blood, serum and serosal fluids, plasma, cerebrospinalfluid, ocular fluids, lymph, urine, saliva, and the like. Tissue samplesmay include samples from tissues, organs or localized regions. Forexample, samples may be derived from particular organs, parts of organs,or fluids or cells within those organs. In certain embodiments, samplesmay be derived from the brain (e.g., whole brain or certain segments ofbrain or certain types of cells in the brain, such as, e.g., neurons andglial cells (astrocytes, oligodendrocytes, microglial cells)). In someembodiments, a “sample derived from a subject” refers to blood or plasmadrawn from the subject. In further embodiments, a “sample derived from asubject” refers to brain tissue (or subcomponents thereof) or retinaltissue (or subcomponents thereof) derived from the subject.

II. RNAi Agents of the Disclosure

Described herein are RNAi agents which inhibit the expression of an APPgene. In one embodiment, the RNAi agent includes double strandedribonucleic acid (dsRNA) molecules for inhibiting the expression of anAPP gene in a cell, such as a cell within a subject, e.g., a mammal,such as a human having an APP-associated disorder, e.g., cerebralamyloid angiopathy (CAA) or Alzheimer's disease (AD), including, e.g.,early onset familial Alzheimer disease (EOFAD). The dsRNA includes anantisense strand having a region of complementarity which iscomplementary to at least a part of an mRNA formed in the expression ofan APP gene, The region of complementarity is about 30 nucleotides orless in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, or 18 nucleotides or less in length). Upon contact with a cellexpressing the APP gene, the RNAi agent inhibits the expression of theAPP gene (e.g., a human, a primate, a non-primate, or a bird APP gene)by at least about 10% as assayed by, for example, a PCR or branched DNA(bDNA)-based method, or by a protein-based method, such as byimmunofluorescence analysis, using, for example, Western Blotting orflowcytometric techniques.

A dsRNA includes two RNA strands that are complementary and hybridize toform a duplex structure under conditions in which the dsRNA will beused. One strand of a dsRNA (the antisense strand) includes a region ofcomplementarity that is substantially complementary, and generally fullycomplementary, to a target sequence. The target sequence can be derivedfrom the sequence of an mRNA formed during the expression of an APPgene. The other strand (the sense strand) includes a region that iscomplementary to the antisense strand, such that the two strandshybridize and form a duplex structure when combined under suitableconditions. As described elsewhere herein and as known in the art, thecomplementary sequences of a dsRNA can also be contained asself-complementary regions of a single nucleic acid molecule, as opposedto being on separate oligonucleotides.

Generally, the duplex structure is between 15 and 30 base pairs inlength, e.g., between, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23,15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27,18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28,19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29,20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29,21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs in length.In certain preferred embodiments, the duplex structure is between 18 and25 base pairs in length, e.g., 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-25, 20-24, 20-23, 20-22,20-21, 21-25, 21-24, 21-23, 21-22, 22-25, 22-24, 22-23, 23-25, 23-24 or24-25 base pairs in length. Ranges and lengths intermediate to the aboverecited ranges and lengths are also contemplated to be part of thedisclosure.

Similarly, the region of complementarity to the target sequence isbetween 15 and 30 nucleotides in length, e.g., between 15-29, 15-28,15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18,15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22,18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23,19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24,20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24,21-23, or 21-22 nucleotides in length. Ranges and lengths intermediateto the above recited ranges and lengths are also contemplated to be partof the disclosure.

In some embodiments, the dsRNA is between about 15 and about 23nucleotides in length, or between about 25 and about 30 nucleotides inlength. In general, the dsRNA is long enough to serve as a substrate forthe Dicer enzyme. For example, it is well known in the art that dsRNAslonger than about 21-23 nucleotides can serve as substrates for Dicer.As the ordinarily skilled person will also recognize, the region of anRNA targeted for cleavage will most often be part of a larger RNAmolecule, often an mRNA molecule. Where relevant, a “part” of an mRNAtarget is a contiguous sequence of an mRNA target of sufficient lengthto allow it to be a substrate for RNAi-directed cleavage (i.e., cleavagethrough a RISC pathway).

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of about 9to 36 base pairs, e.g., about 10-36, 11-36, 12-36, 13-36, 14-36, 15-36,9-35, 10-35, 11-35, 12-35, 13-35, 14-35, 15-35, 9-34, 10-34, 11-34,12-34, 13-34, 14-34, 15-34, 9-33, 10-33, 11-33, 12-33, 13-33, 14-33,15-33, 9-32, 10-32, 11-32, 12-32, 13-32, 14-32, 15-32, 9-31, 10-31,11-31, 12-31, 13-32, 14-31, 15-31, 15-30, 15-29, 15-28, 15-27, 15-26,15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30,18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20,19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21,19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22,20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22base pairs. Thus, in one embodiment, to the extent that it becomesprocessed to a functional duplex, of e.g., 15-30 base pairs, thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, a miRNA is a dsRNA. In another embodiment, a dsRNA is not anaturally occurring miRNA. In another embodiment, a RNAi agent useful totarget APP expression is not generated in the target cell by cleavage ofa larger dsRNA.

A dsRNA as described herein can further include one or moresingle-stranded nucleotide overhangs e.g., 1, 2, 3, or 4 nucleotides.dsRNAs having at least one nucleotide overhang can have unexpectedlysuperior inhibitory properties relative to their blunt-endedcounterparts. A nucleotide overhang can comprise or consist of anucleotide/nucleoside analog, including a deoxynucleotide/nucleoside.The overhang(s) can be on the sense strand, the antisense strand or anycombination thereof. Furthermore, the nucleotide(s) of an overhang canbe present on the 5′-end, 3′-end or both ends of either an antisense orsense strand of a dsRNA.

A dsRNA can be synthesized by standard methods known in the art asfurther discussed below, e.g., by use of an automated DNA synthesizer,such as are commercially available from, for example, Biosearch, AppliedBiosystems, Inc.

RNAi agents of the disclosure may be prepared using a two-stepprocedure. First, the individual strands of the double stranded RNAmolecule are prepared separately. Then, the component strands areannealed. The individual strands of the siRNA compound can be preparedusing solution-phase or solid-phase organic synthesis or both. Organicsynthesis offers the advantage that the oligonucleotide strandscomprising unnatural or modified nucleotides can be easily prepared.Single-stranded oligonucleotides of the disclosure can be prepared usingsolution-phase or solid-phase organic synthesis or both.

In one aspect, a dsRNA of the disclosure includes at least twonucleotide sequences, a sense sequence and an antisense sequence. Thesense strand sequence may be selected from the group of sequencesprovided in any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B,and 26 and the corresponding nucleotide sequence of the antisense strandof the sense strand may be selected from the group of sequences of anyone of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, and 26. In thisaspect, one of the two sequences is complementary to the other of thetwo sequences, with one of the sequences being substantiallycomplementary to a sequence of an mRNA generated in the expression of anAPP gene. As such, in this aspect, a dsRNA will include twooligonucleotides, where one oligonucleotide is described as the sensestrand (passenger strand) in any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9,10-15, 16A, 16B, and 26, and the second oligonucleotide is described asthe corresponding antisense strand (guide strand) of the sense strand inany one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, and 26.Accordingly, by way of example, the following pairwise selections ofsense and antisense strand sequences of Table 3 are expresslycontemplated as forming duplexes of the instant disclosure: SEQ ID NOs:855 and 856; SEQ ID NOs: 857 and 858; SEQ ID NOs: 859 and 860; SEQ IDNOs: 861 and 862; SEQ ID NOs: 863 and 864; SEQ ID NOs: 865 and 866; SEQID NOs: 867 and 868; SEQ ID NOs: 869 and 870; SEQ ID NOs: 871 and 872;SEQ ID NOs: 873 and 874; SEQ ID NOs: 875 and 876; SEQ ID NOs: 877 and878; SEQ ID NOs: 879 and 880; SEQ ID NOs: 881 and 882; SEQ ID NOs: 883and 884; SEQ ID NOs: 885 and 886; SEQ ID NOs: 887 and 888; SEQ ID NOs:889 and 890; SEQ ID NOs: 891 and 892; SEQ ID NOs: 893 and 894; SEQ IDNOs: 895 and 896; SEQ ID NOs: 897 and 898; SEQ ID NOs: 899 and 900; SEQID NOs: 901 and 902; SEQ ID NOs: 903 and 904; SEQ ID NOs: 905 and 906;SEQ ID NOs: 907 and 908; SEQ ID NOs: 909 and 910; SEQ ID NOs: 911 and912; SEQ ID NOs: 913 and 914; SEQ ID NOs: 915 and 916; SEQ ID NOs: 917and 918; SEQ ID NOs: 919 and 920; SEQ ID NOs: 921 and 922; SEQ ID NOs:923 and 924; SEQ ID NOs: 925 and 926; SEQ ID NOs: 927 and 928; SEQ IDNOs: 929 and 930; SEQ ID NOs: 931 and 932; SEQ ID NOs: 933 and 934; SEQID NOs: 935 and 936; SEQ ID NOs: 937 and 938; SEQ ID NOs: 939 and 940;SEQ ID NOs: 941 and 942; SEQ ID NOs: 943 and 944; SEQ ID NOs: 945 and946; SEQ ID NOs: 947 and 948; SEQ ID NOs: 949 and 950; SEQ ID NOs: 951and 952; SEQ ID NOs: 953 and 954; SEQ ID NOs: 955 and 956; SEQ ID NOs:957 and 958; SEQ ID NOs: 959 and 960; SEQ ID NOs: 961 and 962; SEQ IDNOs: 963 and 964; SEQ ID NOs: 965 and 966; SEQ ID NOs: 967 and 968; SEQID NOs: 969 and 970; SEQ ID NOs: 971 and 972; SEQ ID NOs: 973 and 974;SEQ ID NOs: 975 and 976; SEQ ID NOs: 977 and 978; SEQ ID NOs: 979 and980; SEQ ID NOs: 981 and 982; SEQ ID NOs: 983 and 984; SEQ ID NOs: 985and 986; SEQ ID NOs: 987 and 988; SEQ ID NOs: 989 and 990; SEQ ID NOs:991 and 992; SEQ ID NOs: 993 and 994; SEQ ID NOs: 995 and 996; SEQ IDNOs: 997 and 998; SEQ ID NOs: 999 and 1000; SEQ ID NOs: 1001 and 1002;SEQ ID NOs: 1003 and 1004; SEQ ID NOs: 1005 and 1006; SEQ ID NOs: 1007and 1008; SEQ ID NOs: 1009 and 1010; SEQ ID NOs: 1011 and 1012; SEQ IDNOs: 1013 and 1014; SEQ ID NOs: 1015 and 1016; SEQ ID NOs: 1017 and1018; SEQ ID NOs: 1019 and 1020; SEQ ID NOs: 1021 and 1022; SEQ ID NOs:1023 and 1024; SEQ ID NOs: 1025 and 1026; SEQ ID NOs: 1027 and 1028; SEQID NOs: 1029 and 1030; SEQ ID NOs: 1031 and 1032; SEQ ID NOs: 1033 and1034; SEQ ID NOs: 1035 and 1036; SEQ ID NOs: 1037 and 1038; SEQ ID NOs:1039 and 1040; SEQ ID NOs: 1041 and 1042; SEQ ID NOs: 1043 and 1044; SEQID NOs: 1045 and 1046; SEQ ID NOs: 1047 and 1048; SEQ ID NOs: 1049 and1050; SEQ ID NOs: 1051 and 1052; SEQ ID NOs: 1053 and 1054; SEQ ID NOs:1055 and 1056; SEQ ID NOs: 1057 and 1058; SEQ ID NOs: 1059 and 1060; SEQID NOs: 1061 and 1062; SEQ ID NOs: 1063 and 1064; SEQ ID NOs: 1065 and1066; SEQ ID NOs: 1067 and 1068; SEQ ID NOs: 1069 and 1070; SEQ ID NOs:1071 and 1072; SEQ ID NOs: 1073 and 1074; SEQ ID NOs: 1075 and 1076; SEQID NOs: 1077 and 1078; SEQ ID NOs: 1079 and 1080; SEQ ID NOs: 1081 and1082; SEQ ID NOs: 1083 and 1084; SEQ ID NOs: 1085 and 1086; SEQ ID NOs:1087 and 1088; SEQ ID NOs: 1089 and 1090; SEQ ID NOs: 1091 and 1092; SEQID NOs: 1093 and 1094; SEQ ID NOs: 1095 and 1096; SEQ ID NOs: 1097 and1098; SEQ ID NOs: 1099 and 1100; SEQ ID NOs: 1101 and 1102; SEQ ID NOs:1103 and 1104; SEQ ID NOs: 1105 and 1106; SEQ ID NOs: 1107 and 1108; SEQID NOs: 1109 and 1110; SEQ ID NOs: 1111 and 1112; SEQ ID NOs: 1113 and1114; SEQ ID NOs: 1115 and 1116; SEQ ID NOs: 1117 and 1118; SEQ ID NOs:1119 and 1120; SEQ ID NOs: 1121 and 1122; SEQ ID NOs: 1123 and 1124; SEQID NOs: 1125 and 1126; SEQ ID NOs: 1127 and 1128; SEQ ID NOs: 1129 and1130; SEQ ID NOs: 1131 and 1132; SEQ ID NOs: 1133 and 1134; SEQ ID NOs:1135 and 1136; SEQ ID NOs: 1137 and 1138; SEQ ID NOs: 1139 and 1140; SEQID NOs: 1141 and 1142; SEQ ID NOs: 1143 and 1144; SEQ ID NOs: 1145 and1146; SEQ ID NOs: 1147 and 1148; SEQ ID NOs: 1149 and 1150; SEQ ID NOs:1151 and 1152; SEQ ID NOs: 1153 and 1154; SEQ ID NOs: 1155 and 1156; SEQID NOs: 1157 and 1158; SEQ ID NOs: 1159 and 1160; SEQ ID NOs: 1161 and1162; SEQ ID NOs: 1163 and 1164; SEQ ID NOs: 1165 and 1166; SEQ ID NOs:1167 and 1168; SEQ ID NOs: 1169 and 1170; SEQ ID NOs: 1171 and 1172; SEQID NOs: 1173 and 1174; SEQ ID NOs: 1175 and 1176; SEQ ID NOs: 1177 and1178; SEQ ID NOs: 1179 and 1180; SEQ ID NOs: 1181 and 1182; SEQ ID NOs:1183 and 1184; SEQ ID NOs: 1185 and 1186; SEQ ID NOs: 1187 and 1188; SEQID NOs: 1189 and 1190; SEQ ID NOs: 1191 and 1192; SEQ ID NOs: 1193 and1194; SEQ ID NOs: 1195 and 1196; SEQ ID NOs: 1197 and 1198; SEQ ID NOs:1199 and 1200; SEQ ID NOs: 1201 and 1202; SEQ ID NOs: 1203 and 1204; SEQID NOs: 1205 and 1206; SEQ ID NOs: 1207 and 1208; SEQ ID NOs: 1209 and1210; SEQ ID NOs: 1211 and 1212; SEQ ID NOs: 1213 and 1214; SEQ ID NOs:1215 and 1216; SEQ ID NOs: 1217 and 1218; SEQ ID NOs: 1219 and 1220; SEQID NOs: 1221 and 1222; SEQ ID NOs: 1223 and 1224; SEQ ID NOs: 1225 and1226; SEQ ID NOs: 1227 and 1228; SEQ ID NOs: 1229 and 1230; SEQ ID NOs:1231 and 1232; SEQ ID NOs: 1233 and 1234; SEQ ID NOs: 1235 and 1236; SEQID NOs: 1237 and 1238; SEQ ID NOs: 1239 and 1240; SEQ ID NOs: 1241 and1242; SEQ ID NOs: 1243 and 1244; SEQ ID NOs: 1245 and 1246; SEQ ID NOs:1247 and 1248; SEQ ID NOs: 1249 and 1250; SEQ ID NOs: 1251 and 1252; SEQID NOs: 1253 and 1254; SEQ ID NOs: 1255 and 1256; SEQ ID NOs: 1257 and1258; SEQ ID NOs: 1259 and 1260; SEQ ID NOs: 1261 and 1262; SEQ ID NOs:1263 and 1264; SEQ ID NOs: 1265 and 1266; SEQ ID NOs: 1267 and 1268; SEQID NOs: 1269 and 1270; SEQ ID NOs: 1271 and 1272; SEQ ID NOs: 1273 and1274; SEQ ID NOs: 1275 and 1276; SEQ ID NOs: 1277 and 1278; SEQ ID NOs:1279 and 1280; SEQ ID NOs: 1281 and 1282; SEQ ID NOs: 1283 and 1284; SEQID NOs: 1285 and 1286; SEQ ID NOs: 1287 and 1288; SEQ ID NOs: 1289 and1290; SEQ ID NOs: 1291 and 1292; SEQ ID NOs: 1293 and 1294; SEQ ID NOs:1295 and 1296; SEQ ID NOs: 1297 and 1298; SEQ ID NOs: 1299 and 1300; SEQID NOs: 1301 and 1302; SEQ ID NOs: 1303 and 1304; SEQ ID NOs: 1305 and1306; SEQ ID NOs: 1307 and 1308; SEQ ID NOs: 1309 and 1310; SEQ ID NOs:1311 and 1312; SEQ ID NOs: 1313 and 1314; SEQ ID NOs: 1315 and 1316; SEQID NOs: 1317 and 1318; SEQ ID NOs: 1319 and 1320; SEQ ID NOs: 1321 and1322; SEQ ID NOs: 1323 and 1324; SEQ ID NOs: 1325 and 1326; SEQ ID NOs:1327 and 1328; SEQ ID NOs: 1329 and 1330; SEQ ID NOs: 1331 and 1332; SEQID NOs: 1333 and 1334; SEQ ID NOs: 1335 and 1336; SEQ ID NOs: 1337 and1338; SEQ ID NOs: 1339 and 1340; SEQ ID NOs: 1341 and 1342; SEQ ID NOs:1343 and 1344; SEQ ID NOs: 1345 and 1346; SEQ ID NOs: 1347 and 1348; SEQID NOs: 1349 and 1350; SEQ ID NOs: 1351 and 1352; SEQ ID NOs: 1353 and1354; SEQ ID NOs: 1355 and 1356; SEQ ID NOs: 1357 and 1358; SEQ ID NOs:1359 and 1360; SEQ ID NOs: 1361 and 1362; SEQ ID NOs: 1363 and 1364; SEQID NOs: 1365 and 1366; SEQ ID NOs: 1367 and 1368; SEQ ID NOs: 1369 and1370; SEQ ID NOs: 1371 and 1372; SEQ ID NOs: 1373 and 1374; SEQ ID NOs:1375 and 1376; SEQ ID NOs: 1377 and 1378; SEQ ID NOs: 1379 and 1380; SEQID NOs: 1381 and 1382; SEQ ID NOs: 1383 and 1384; SEQ ID NOs: 1385 and1386; SEQ ID NOs: 1387 and 1388; SEQ ID NOs: 1389 and 1390; SEQ ID NOs:1391 and 1392; SEQ ID NOs: 1393 and 1394; SEQ ID NOs: 1395 and 1396; SEQID NOs: 1397 and 1398; SEQ ID NOs: 1399 and 1400; and SEQ ID NOs: 1401and 1402. Similarly, pairwise combinations of sense and antisensestrands of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, and 26 ofthe instant disclosure are also expressly contemplated, including, e.g.,a sense strand selected from Table 2A together with an antisense strandselected from Table 2B, or vice versa, etc.

In one embodiment, the substantially complementary sequences of thedsRNA are contained on separate oligonucleotides. In another embodiment,the substantially complementary sequences of the dsRNA are contained ona single oligonucleotide.

It will be understood that, although the sequences in Tables 2A, 2B, 5A,5B, 9, 10, 12, 14, 16A, 16B, and 26 are described as modified and/orconjugated sequences, the RNA of the RNAi agent of the disclosure e.g.,a dsRNA of the disclosure, may comprise any one of the sequences setforth in any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, and26 that is un-modified, un-conjugated, and/or modified and/or conjugateddifferently than described therein.

The skilled person is well aware that dsRNAs having a duplex structureof between about 20 and 23 base pairs, e.g., 21, base pairs have beenhailed as particularly effective in inducing RNA interference (Elbashiret al., (2001) EMBO 1, 20:6877-6888). However, others have found thatshorter or longer RNA duplex structures can also be effective (Chu andRana (2007) RNA 14:1714-1719; Kim et al. (2005) Nat Biotech 23:222-226).In the embodiments described above, by virtue of the nature of theoligonucleotide sequences provided herein, dsRNAs described herein caninclude at least one strand of a length of minimally 21 nucleotides. Itcan be reasonably expected that shorter duplexes minus only a fewnucleotides on one or both ends can be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a sequence of atleast 15, 16, 17, 18, 19, 20, or more contiguous nucleotides derivedfrom one of the sequences provided herein, and differing in theirability to inhibit the expression of an APP gene by not more than about5, 10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the fullsequence, are contemplated to be within the scope of the presentdisclosure.

In addition, the RNAs described herein identify a site(s) in an APPtranscript that is susceptible to RISC-mediated cleavage. As such, thepresent disclosure further features RNAi agents that target within thissite(s). As used herein, a RNAi agent is said to target within aparticular site of an RNA transcript if the RNAi agent promotes cleavageof the transcript anywhere within that particular site. Such a RNAiagent will generally include at least about 15 contiguous nucleotidesfrom one of the sequences provided herein coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in an APP gene.

A RNAi agent as described herein can contain one or more mismatches tothe target sequence. In one embodiment, a RNAi agent as described hereincontains no more than 3 mismatches. In certain embodiments, if theantisense strand of the RNAi agent contains mismatches to the targetsequence, the mismatch can optionally be restricted to be within thelast 5 nucleotides from either the 5′- or 3′-end of the region ofcomplementarity. For example, in such embodiments, for a 23 nucleotideRNAi agent, the strand which is complementary to a region of an APPgene, generally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether a RNAi agent containing a mismatch to atarget sequence is effective in inhibiting the expression of an APPgene. Consideration of the efficacy of RNAi agents with mismatches ininhibiting expression of an APP gene is important, especially if theparticular region of complementarity in an APP gene is known to havepolymorphic sequence variation within the population.

III. Modified RNAi Agents of the Disclosure

In one embodiment, the RNA of the RNAi agent of the disclosure e.g., adsRNA, is un-modified, and does not comprise, e.g., chemicalmodifications and/or conjugations known in the art and described herein.In another embodiment, the RNA of a RNAi agent of the disclosure, e.g.,a dsRNA, is chemically modified to enhance stability or other beneficialcharacteristics. In certain embodiments of the disclosure, substantiallyall of the nucleotides of a RNAi agent of the disclosure are modified.In other embodiments of the disclosure, all of the nucleotides of a RNAiagent of the disclosure are modified. RNAi agents of the disclosure inwhich “substantially all of the nucleotides are modified” are largelybut not wholly modified and can include not more than 5, 4, 3, 2, or 1unmodified nucleotides. In still other embodiments of the disclosure,RNAi agents of the disclosure can include not more than 5, 4, 3, 2 or 1modified nucleotides.

The nucleic acids featured in the disclosure can be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,end modifications, e.g., 5′-end modifications (phosphorylation,conjugation, inverted linkages) or 3′-end modifications (conjugation,DNA nucleotides, inverted linkages, etc.); base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases; sugar modifications (e.g., atthe 2′-position or 4′-position) or replacement of the sugar; and/orbackbone modifications, including modification or replacement of thephosphodiester linkages. Specific examples of RNAi agents useful in theembodiments described herein include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In some embodiments, amodified RNAi agent will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′-linked analogs ofthese, and those having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat.RE39464, the entire contents of each of which are hereby incorporatedherein by reference.

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, the entire contents of each of which are hereby incorporatedherein by reference.

In other embodiments, suitable RNA mimetics are contemplated for use inRNAi agents, in which both the sugar and the internucleoside linkage,i.e., the backbone, of the nucleotide units are replaced with novelgroups. The base units are maintained for hybridization with anappropriate nucleic acid target compound. One such oligomeric compound,an RNA mimetic that has been shown to have excellent hybridizationproperties, is referred to as a peptide nucleic acid (PNA). In PNAcompounds, the sugar backbone of an RNA is replaced with an amidecontaining backbone, in particular an aminoethylglycine backbone. Thenucleobases are retained and are bound directly or indirectly to azanitrogen atoms of the amide portion of the backbone. Representative U.S.patents that teach the preparation of PNA compounds include, but are notlimited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, theentire contents of each of which are hereby incorporated herein byreference. Additional PNA compounds suitable for use in the RNAi agentsof the disclosure are described in, for example, in Nielsen et al.,Science, 1991, 254, 1497-1500.

Some embodiments featured in the disclosure include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular—CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂—[known as amethylene (methylimino) or MMI backbone], —CH₂—N(CH₃)—N(CH₃)—CH₂— and—N(CH₃)—CH₂—CH₂—[wherein the native phosphodiester backbone isrepresented as —O—P—O—CH₂—] of the above-referenced U.S. Pat. No.5,489,677, and the amide backbones of the above-referenced U.S. Pat. No.5,602,240. In some embodiments, the RNAs featured herein have morpholinobackbone structures of the above-referenced U.S. Pat. No. 5,034,506.

Modified RNAs can also contain one or more substituted sugar moieties.The RNAi agents, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′-position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl can be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(n)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]2, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aRNAi agent, or a group for improving the pharmacodynamic properties of aRNAi agent, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Helv. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-O-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂. Further exemplary modifications include:5′-Me-2′-F nucleotides, 5′-Me-2′-OMe nucleotides,5′-Me-2′-deoxynucleotides, (both R and S isomers in these threefamilies); 2′-alkoxyalkyl; and 2′-NMA (N-methylacetamide).

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂), 2′-O-hexadecyl, and 2′-fluoro (2′-F). Similarmodifications can also be made at other positions on the RNA of a RNAiagent, particularly the 3′ position of the sugar on the 3′ terminalnucleotide or in 2′-5′ linked dsRNAs and the 5′ position of 5′ terminalnucleotide. RNAi agents can also have sugar mimetics such as cyclobutylmoieties in place of the pentofuranosyl sugar. Representative U.S.patents that teach the preparation of such modified sugar structuresinclude, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800;5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785;5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300;5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920,certain of which are commonly owned with the instant application. Theentire contents of each of the foregoing are hereby incorporated hereinby reference.

A RNAi agent of the disclosure can also include nucleobase (oftenreferred to in the art simply as “base”) modifications or substitutions.As used herein, “unmodified” or “natural” nucleobases include the purinebases adenine (A) and guanine (G), and the pyrimidine bases thymine (T),cytosine (C) and uracil (U). Modified nucleobases include othersynthetic and natural nucleobases such as 5-methylcytosine (5-me-C),5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine,6-methyl and other alkyl derivatives of adenine and guanine, 2-propyland other alkyl derivatives of adenine and guanine, 2-thiouracil,2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyluracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil(pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl,8-hydroxyl anal other 8-substituted adenines and guanines, 5-halo,particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracilsand cytosines, 7-methylguanine and 7-methyladenine, 8-azaguanine and8-azaadenine, 7-deazaguanine and 7-daazaadenine and 3-deazaguanine and3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat.No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry,Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008; thosedisclosed in The Concise Encyclopedia Of Polymer Science AndEngineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons,1990, these disclosed by Englisch et al., (1991) Angewandte Chemie,International Edition, 30:613, and those disclosed by Sanghvi, Y S.,Chapter 15, dsRNA Research and Applications, pages 289-302, Crooke, S.T. and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobasesare particularly useful for increasing the binding affinity of theoligomeric compounds featured in the disclosure. These include5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6substituted purines, including 2-aminopropyladenine, 5-propynyluraciland 5-propynylcytosine. 5-methylcytosine substitutions have been shownto increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y.S., Crooke, S. T. and Lebleu, B., Eds., dsRNA Research and Applications,CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary basesubstitutions, even more particularly when combined with2′-O-methoxyethyl sugar modifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. Nos.3,687,808, 4,845,205; 5,130,30; 5,134,066; 5,175,273; 5,367,066;5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711;5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941;5,750,692; 6,015,886; 6,147,200; 6,166,197; 6,222,025; 6,235,887;6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610; 7,427,672; and7,495,088, the entire contents of each of which are hereby incorporatedherein by reference.

A RNAi agent of the disclosure can also be modified to include one ormore locked nucleic acids (LNA). A locked nucleic acid is a nucleotidehaving a modified ribose moiety in which the ribose moiety comprises anextra bridge connecting the 2′ and 4′ carbons. This structureeffectively “locks” the ribose in the 3′-endo structural conformation.The addition of locked nucleic acids to siRNAs has been shown toincrease siRNA stability in serum, and to reduce off-target effects(Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al.,(2003) Nucleic Acids Research 31(12):3185-3193).

A RNAi agent of the disclosure can also be modified to include one ormore bicyclic sugar moities. A “bicyclic sugar” is a furanosyl ringmodified by the bridging of two atoms. A “bicyclic nucleoside” (“BNA”)is a nucleoside having a sugar moiety comprising a bridge connecting twocarbon atoms of the sugar ring, thereby forming a bicyclic ring system.In certain embodiments, the bridge connects the 4′-carbon and the2′-carbon of the sugar ring. Thus, in some embodiments an agent of thedisclosure may include one or more locked nucleic acids (LNA). A lockednucleic acid is a nucleotide having a modified ribose moiety in whichthe ribose moiety comprises an extra bridge connecting the 2′ and 4′carbons. In other words, an LNA is a nucleotide comprising a bicyclicsugar moiety comprising a 4′-CH₂—O-2′ bridge. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193). Examples of bicyclic nucleosides for use inthe polynucleotides of the disclosure include without limitationnucleosides comprising a bridge between the 4′ and the 2′ ribosyl ringatoms. In certain embodiments, the antisense polynucleotide agents ofthe disclosure include one or more bicyclic nucleosides comprising a 4′to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides,include but are not limited to 4′-(CH2)-O-2′ (LNA); 4′-(CH2)-S-2;4′-(CH2)2-O-2′ (ENA); 4′-CH(CH3)-O-2′ (also referred to as “constrainedethyl” or “cEt”) and 4′-CH(CH2OCH3)-O-2′ (and analogs thereof; see,e.g., U.S. Pat. No. 7,399,845); 4′-C(CH3)(CH3)-O-2′ (and analogsthereof; see e.g., U.S. Pat. No. 8,278,283); 4′-CH2-N(OCH3)-2′ (andanalogs thereof; see e.g., U.S. Pat. No. 8,278,425); 4′-CH2-O—N(CH3)-2′(see, e.g., U.S. Patent Publication No. 2004/0171570); 4′-CH2-N(R)—O-2′,wherein R is H, C1-C12 alkyl, or a protecting group (see, e.g., U.S.Pat. No. 7,427,672); 4′-CH2-C(H)(CH3)-2′ (see, e.g., Chattopadhyaya etal., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2-C(═CH2)-2′ (andanalogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entirecontents of each of the foregoing are hereby incorporated herein byreference.

Additional representative U.S. patents and US patent Publications thatteach the preparation of locked nucleic acid nucleotides include, butare not limited to, the following: U.S. Pat. Nos. 6,268,490; 6,525,191;6,670,461; 6,770,748; 6,794,499; 6,998,484; 7,053,207; 7,034,133;7,084,125; 7,399,845; 7,427,672; 7,569,686; 7,741,457; 8,022,193;8,030,467; 8,278,425; 8,278,426; 8,278,283; US 2008/0039618; and US2009/0012281, the entire contents of each of which are herebyincorporated herein by reference.

Any of the foregoing bicyclic nucleosides can be prepared having one ormore stereochemical sugar configurations including for exampleα-L-ribofuranose and β-D-ribofuranose (see WO 99/14226).

A RNAi agent of the disclosure can also be modified to include one ormore constrained ethyl nucleotides. As used herein, a “constrained ethylnucleotide” or “cEt” is a locked nucleic acid comprising a bicyclicsugar moiety comprising a 4′-CH(CH3)-O-2′ bridge. In one embodiment, aconstrained ethyl nucleotide is in the S conformation referred to hereinas “S-cEt.”

A RNAi agent of the disclosure may also include one or more“conformationally restricted nucleotides” (“CRN”). CRN are nucleotideanalogs with a linker connecting the C2′ and C4′ carbons of ribose orthe C3 and —C5′ carbons of ribose. CRN lock the ribose ring into astable conformation and increase the hybridization affinity to mRNA. Thelinker is of sufficient length to place the oxygen in an optimalposition for stability and affinity resulting in less ribose ringpuckering.

Representative publications that teach the preparation of certain of theabove noted CRN include, but are not limited to, US Patent PublicationNo. 2013/0190383; and PCT publication WO 2013/036868, the entirecontents of each of which are hereby incorporated herein by reference.

In some embodiments, a RNAi agent of the disclosure comprises one ormore monomers that are UNA (unlocked nucleic acid) nucleotides. UNA isunlocked acyclic nucleic acid, wherein any of the bonds of the sugar hasbeen removed, forming an unlocked “sugar” residue. In one example, UNAalso encompasses monomer with bonds between C1′-C4′ have been removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar hasbeen removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) andFluiter et al., Mol. Biosyst., 2009, 10, 1039 hereby incorporated byreference).

Representative U.S. publications that teach the preparation of UNAinclude, but are not limited to, U.S. Pat. No. 8,314,227; and US PatentPublication Nos. 2013/0096289; 2013/0011922; and 2011/0313020, theentire contents of each of which are hereby incorporated herein byreference.

Potentially stabilizing modifications to the ends of RNA molecules caninclude N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc),N-(caproyl-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol(Hyp-NHAc), thymidine-2′-O-deoxythymidine (ether),N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino),2-docosanoyl-uridine-3″-phosphate, inverted base dT(idT) and others.Disclosure of this modification can be found in PCT Publication No. WO2011/005861.

Other modifications of a RNAi agent of the disclosure include a 5′phosphate or 5′ phosphate mimic, e.g., a 5′-terminal phosphate orphosphate mimic on the antisense strand of a RNAi agent. Suitablephosphate mimics are disclosed in, for example US Patent Publication No.2012/0157511, the entire contents of which are incorporated herein byreference.

A. Modified RNAi Agents Comprising Motifs of the Disclosure

In certain aspects of the disclosure, the double-stranded RNAi agents ofthe disclosure include agents with chemical modifications as disclosed,for example, in WO 2013/075035, filed on Nov. 16, 2012, the entirecontents of which are incorporated herein by reference. As shown hereinand in PCT Publication No. WO 2013/075035, a superior result may beobtained by introducing one or more motifs of three identicalmodifications on three consecutive nucleotides into a sense strandand/or antisense strand of an RNAi agent, particularly at or near thecleavage site. In some embodiments, the sense strand and antisensestrand of the RNAi agent may otherwise be completely modified. Theintroduction of these motifs interrupts the modification pattern, ifpresent, of the sense and/or antisense strand. The RNAi agent may beoptionally conjugated with a C16 ligand, for instance on the sensestrand. The RNAi agent may be optionally modified with a (S)-glycolnucleic acid (GNA) modification, for instance on one or more residues ofthe antisense strand. The resulting RNAi agents present superior genesilencing activity.

More specifically, it has been surprisingly discovered that when thesense strand and antisense strand of the double-stranded RNAi agent arecompletely modified to have one or more motifs of three identicalmodifications on three consecutive nucleotides at or near the cleavagesite of at least one strand of an RNAi agent, the gene silencingactivity of the RNAi agent was superiorly enhanced.

Accordingly, the disclosure provides double stranded RNAi agents capableof inhibiting the expression of a target gene (i.e., an APP gene) invivo. The RNAi agent comprises a sense strand and an antisense strand.Each strand of the RNAi agent may range from 12-30 nucleotides inlength. For example, each strand may be between 14-30 nucleotides inlength, 17-30 nucleotides in length, 25-30 nucleotides in length, 27-30nucleotides in length, 17-23 nucleotides in length, 17-21 nucleotides inlength, 17-19 nucleotides in length, 19-25 nucleotides in length, 19-23nucleotides in length, 19-21 nucleotides in length, 21-25 nucleotides inlength, or 21-23 nucleotides in length.

The sense strand and antisense strand typically form a duplex doublestranded RNA (“dsRNA”), also referred to herein as an “RNAi agent.” Theduplex region of an RNAi agent may be 12-30 nucleotide pairs in length.For example, the duplex region can be between 14-30 nucleotide pairs inlength, 17-30 nucleotide pairs in length, 27-30 nucleotide pairs inlength, 17-23 nucleotide pairs in length, 17-21 nucleotide pairs inlength, 17-19 nucleotide pairs in length, 19-25 nucleotide pairs inlength, 19-23 nucleotide pairs in length, 19-21 nucleotide pairs inlength, 21-25 nucleotide pairs in length, or 21-23 nucleotide pairs inlength. In another example, the duplex region is selected from 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, and 27 nucleotides in length.

In one embodiment, the RNAi agent may contain one or more overhangregions and/or capping groups at the 3′-end, 5′-end, or both ends of oneor both strands. The overhang can be 1-6 nucleotides in length, forinstance 2-6 nucleotides in length, 1-5 nucleotides in length, 2-5nucleotides in length, 1-4 nucleotides in length, 2-4 nucleotides inlength, 1-3 nucleotides in length, 2-3 nucleotides in length, or 1-2nucleotides in length. The overhangs can be the result of one strandbeing longer than the other, or the result of two strands of the samelength being staggered. The overhang can form a mismatch with the targetmRNA or it can be complementary to the gene sequences being targeted orcan be another sequence. The first and second strands can also bejoined, e.g., by additional bases to form a hairpin, or by othernon-base linkers.

In one embodiment, the nucleotides in the overhang region of the RNAiagent can each independently be a modified or unmodified nucleotideincluding, but no limited to 2′-sugar modified, such as, 2-F,2′-Omethyl, thymidine (T), and any combinations thereof.

For example, TT can be an overhang sequence for either end on eitherstrand. The overhang can form a mismatch with the target mRNA or it canbe complementary to the gene sequences being targeted or can be anothersequence.

The 5′- or 3′-overhangs at the sense strand, antisense strand or bothstrands of the RNAi agent may be phosphorylated. In some embodiments,the overhang region(s) contains two nucleotides having aphosphorothioate between the two nucleotides, where the two nucleotidescan be the same or different. In one embodiment, the overhang is presentat the 3′-end of the sense strand, antisense strand, or both strands. Inone embodiment, this 3′-overhang is present in the antisense strand. Inone embodiment, this 3′-overhang is present in the sense strand.

The RNAi agent may contain only a single overhang, which can strengthenthe interference activity of the RNAi, without affecting its overallstability. For example, the single-stranded overhang may be located atthe 3′-terminal end of the sense strand or, alternatively, at the3′-terminal end of the antisense strand. The RNAi may also have a bluntend, located at the 5′-end of the antisense strand (or the 3′-end of thesense strand) or vice versa. Generally, the antisense strand of the RNAihas a nucleotide overhang at the 3′-end, and the 5′-end is blunt. Whilenot wishing to be bound by theory, the asymmetric blunt end at the5′-end of the antisense strand and 3′-end overhang of the antisensestrand favor the guide strand loading into RISC process.

In one embodiment, the RNAi agent is a double ended bluntmer of 19nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 7, 8, 9 from the 5′end. The antisense strand contains at leastone motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In another embodiment, the RNAi agent is a double ended bluntmer of 20nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 8, 9, 10 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In yet another embodiment, the RNAi agent is a double ended bluntmer of21 nucleotides in length, wherein the sense strand contains at least onemotif of three 2′-F modifications on three consecutive nucleotides atpositions 9, 10, 11 from the 5′ end. The antisense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at positions 11, 12, 13 from the 5′ end.

In one embodiment, the RNAi agent comprises a 21 nucleotide sense strandand a 23 nucleotide antisense strand, wherein the sense strand containsat least one motif of three 2′-F modifications on three consecutivenucleotides at positions 9, 10, 11 from the 5′ end; the antisense strandcontains at least one motif of three 2′-O-methyl modifications on threeconsecutive nucleotides at positions 11, 12, 13 from the 5′ end, whereinone end of the RNAi agent is blunt, while the other end comprises a 2nucleotide overhang. Preferably, the 2 nucleotide overhang is at the3′-end of the antisense strand. When the 2 nucleotide overhang is at the3′-end of the antisense strand, there may be two phosphorothioateinternucleotide linkages between the terminal three nucleotides, whereintwo of the three nucleotides are the overhang nucleotides, and the thirdnucleotide is a paired nucleotide next to the overhang nucleotide. Inone embodiment, the RNAi agent additionally has two phosphorothioateinternucleotide linkages between the terminal three nucleotides at boththe 5′-end of the sense strand and at the 5′-end of the antisensestrand. In one embodiment, every nucleotide in the sense strand and theantisense strand of the RNAi agent, including the nucleotides that arepart of the motifs are modified nucleotides. In one embodiment eachresidue is independently modified with a 2′-O-methyl or 3′-fluoro, e.g.,in an alternating motif. Optionally, the RNAi agent further comprises aligand (optionally a C16 ligand).

In one embodiment, the RNAi agent comprises a sense and an antisensestrand, wherein the sense strand is 25-30 nucleotide residues in length,wherein starting from the 5′ terminal nucleotide (position 1) positions1 to 23 of the first strand comprise at least 8 ribonucleotides; theantisense strand is 36-66 nucleotide residues in length and, startingfrom the 3′ terminal nucleotide, comprises at least 8 ribonucleotides inthe positions paired with positions 1-23 of sense strand to form aduplex; wherein at least the 3 ‘ terminal nucleotide of antisense strandis unpaired with sense strand, and up to 6 consecutive 3’ terminalnucleotides are unpaired with sense strand, thereby forming a 3′ singlestranded overhang of 1-6 nucleotides; wherein the 5′ terminus ofantisense strand comprises from 10-30 consecutive nucleotides which areunpaired with sense strand, thereby forming a 10-30 nucleotide singlestranded 5′ overhang; wherein at least the sense strand 5′ terminal and3′ terminal nucleotides are base paired with nucleotides of antisensestrand when sense and antisense strands are aligned for maximumcomplementarity, thereby forming a substantially duplexed region betweensense and antisense strands; and antisense strand is sufficientlycomplementary to a target RNA along at least 19 ribonucleotides ofantisense strand length to reduce target gene expression when the doublestranded nucleic acid is introduced into a mammalian cell; and whereinthe sense strand contains at least one motif of three 2′-F modificationson three consecutive nucleotides, where at least one of the motifsoccurs at or near the cleavage site. The anti sense strand contains atleast one motif of three 2′-O-methyl modifications on three consecutivenucleotides at or near the cleavage site.

In one embodiment, the RNAi agent comprises sense and antisense strands,wherein the RNAi agent comprises a first strand having a length which isat least 25 and at most 29 nucleotides and a second strand having alength which is at most 30 nucleotides with at least one motif of three2′-O-methyl modifications on three consecutive nucleotides at position11, 12, 13 from the 5′ end; wherein the 3′ end of the first strand andthe 5′ end of the second strand form a blunt end and the second strandis 1-4 nucleotides longer at its 3′ end than the first strand, whereinthe duplex region region which is at least 25 nucleotides in length, andthe second strand is sufficiently complemenatary to a target mRNA alongat least 19 nucleotide of the second strand length to reduce target geneexpression when the RNAi agent is introduced into a mammalian cell, andwherein dicer cleavage of the RNAi agent preferentially results in ansiRNA comprising the 3′ end of the second strand, thereby reducingexpression of the target gene in the mammal. Optionally, the RNAi agentfurther comprises a ligand.

In one embodiment, the sense strand of the RNAi agent contains at leastone motif of three identical modifications on three consecutivenucleotides, where one of the motifs occurs at the cleavage site in thesense strand.

In one embodiment, the antisense strand of the RNAi agent can alsocontain at least one motif of three identical modifications on threeconsecutive nucleotides, where one of the motifs occurs at or near thecleavage site in the antisense strand.

For an RNAi agent having a duplex region of 17-23 nucleotide in length,the cleavage site of the antisense strand is typically around the 10, 11and 12 positions from the 5′-end. Thus the motifs of three identicalmodifications may occur at the 9, 10, 11 positions; 10, 11, 12positions; 11, 12, 13 positions; 12, 13, 14 positions; or 13, 14, 15positions of the antisense strand, the count starting from the 1^(st)nucleotide from the 5′-end of the antisense strand, or, the countstarting from the 1^(st) paired nucleotide within the duplex region fromthe 5′-end of the antisense strand. The cleavage site in the antisensestrand may also change according to the length of the duplex region ofthe RNAi from the 5′-end.

The sense strand of the RNAi agent may contain at least one motif ofthree identical modifications on three consecutive nucleotides at thecleavage site of the strand; and the antisense strand may have at leastone motif of three identical modifications on three consecutivenucleotides at or near the cleavage site of the strand. When the sensestrand and the antisense strand form a dsRNA duplex, the sense strandand the antisense strand can be so aligned that one motif of the threenucleotides on the sense strand and one motif of the three nucleotideson the antisense strand have at least one nucleotide overlap, i.e., atleast one of the three nucleotides of the motif in the sense strandforms a base pair with at least one of the three nucleotides of themotif in the antisense strand. Alternatively, at least two nucleotidesmay overlap, or all three nucleotides may overlap.

In one embodiment, the sense strand of the RNAi agent may contain morethan one motif of three identical modifications on three consecutivenucleotides. The first motif may occur at or near the cleavage site ofthe strand and the other motifs may be a wing modification. The term“wing modification” herein refers to a motif occurring at anotherportion of the strand that is separated from the motif at or near thecleavage site of the same strand. The wing modification is eitheradajacent to the first motif or is separated by at least one or morenucleotides. When the motifs are immediately adjacent to each other thenthe chemistry of the motifs are distinct from each other and when themotifs are separated by one or more nucleotide than the chemistries canbe the same or different. Two or more wing modifications may be present.For instance, when two wing modifications are present, each wingmodification may occur at one end relative to the first motif which isat or near cleavage site or on either side of the lead motif.

Like the sense strand, the antisense strand of the RNAi agent maycontain more than one motifs of three identical modifications on threeconsecutive nucleotides, with at least one of the motifs occurring at ornear the cleavage site of the strand. This antisense strand may alsocontain one or more wing modifications in an alignment similar to thewing modifications that may be present on the sense strand.

In one embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two terminal nucleotides at the 3′-end, 5′-end or both ends ofthe strand.

In another embodiment, the wing modification on the sense strand orantisense strand of the RNAi agent typically does not include the firstone or two paired nucleotides within the duplex region at the 3′-end,5′-end or both ends of the strand.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least one wing modification, the wing modifications may fallon the same end of the duplex region, and have an overlap of one, two orthree nucleotides.

When the sense strand and the antisense strand of the RNAi agent eachcontain at least two wing modifications, the sense strand and theantisense strand can be so aligned that two modifications each from onestrand fall on one end of the duplex region, having an overlap of one,two or three nucleotides; two modifications each from one strand fall onthe other end of the duplex region, having an overlap of one, two orthree nucleotides; two modifications one strand fall on each side of thelead motif, having an overlap of one, two or three nucleotides in theduplex region.

In one embodiment, the RNAi agent comprises mismatch(es) with thetarget, within the duplex, or combinations thereof. The mistmatch mayoccur in the overhang region or the duplex region. The base pair may beranked on the basis of their propensity to promote dissociation ormelting (e.g., on the free energy of association or dissociation of aparticular pairing, the simplest approach is to examine the pairs on anindividual pair basis, though next neighbor or similar analysis can alsobe used). In terms of promoting dissociation: A:U is preferred over G:C;G:U is preferred over G:C; and I:C is preferred over G:C (I=inosine).Mismatches, e.g., non-canonical or other than canonical pairings (asdescribed elsewhere herein) are preferred over canonical (A:T, A:U, G:C)pairings; and pairings which include a universal base are preferred overcanonical pairings.

In one embodiment, the RNAi agent comprises at least one of the first 1,2, 3, 4, or 5 base pairs within the duplex regions from the 5′-end ofthe antisense strand independently selected from the group of: A:U, G:U,I:C, and mismatched pairs, e.g., non-canonical or other than canonicalpairings or pairings which include a universal base, to promote thedissociation of the antisense strand at the 5′-end of the duplex.

In one embodiment, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

In another embodiment, the nucleotide at the 3′-end of the sense strandis deoxy-thymine (dT). In another embodiment, the nucleotide at the3′-end of the antisense strand is deoxy-thymine (dT). In one embodiment,there is a short sequence of deoxy-thymine nucleotides, for example, twodT nucleotides on the 3′-end of the sense and/or antisense strand.

In one embodiment, the sense strand sequence may be represented byformula (I):5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′  (I)

wherein:

i and j are each independently 0 or 1;

p and q are each independently 0-6;

each N_(a) independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b) independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p) and n_(q) independently represent an overhang nucleotide;

wherein Nb and Y do not have the same modification; and

XXX, YYY and ZZZ each independently represent one motif of threeidentical modifications on three consecutive nucleotides. Preferably YYYis all 2′-F modified nucleotides.

In one embodiment, the N_(a) and/or N_(b) comprise modifications ofalternating pattern.

In one embodiment, the YYY motif occurs at or near the cleavage site ofthe sense strand. For example, when the RNAi agent has a duplex regionof 17-23 nucleotides in length, the YYY motif can occur at or thevicinity of the cleavage site (e.g.: can occur at positions 6, 7, 8, 7,8, 9, 8, 9, 10, 9, 10, 11, 10, 11, 12 or 11, 12, 13) of—the sensestrand, the count starting from the 1^(st) nucleotide, from the 5′-end;or optionally, the count starting at the 1^(st) paired nucleotide withinthe duplex region, from the 5′-end.

In one embodiment, i is 1 and j is 0, or i is 0 and j is 1, or both iand j are 1. The sense strand can therefore be represented by thefollowing formulas:5′n _(p)-N _(a)-YYY—N _(b)-ZZZ—N _(a)-n _(q)3′  (Ib);5′n _(p)-N _(a)-XXX—N _(b)-YYY—N _(a)-n _(q)3′  (Ic); or5′n _(p)-N _(a)-XXX—N _(b)-YYY—N _(b)-ZZZ—N _(a)-n _(q)3′  (Id).

When the sense strand is represented by formula (Ib), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides.

Each N_(a) independently can represent an oligonucleotide sequencecomprising 2-20, 2-15, or 2-10 modified nucleotides.

When the sense strand is represented as formula (Ic), N_(b) representsan oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7, 0-5, 0-4,0-2 or 0 modified nucleotides. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

When the sense strand is represented as formula (Id), each N_(b)independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Preferably, N_(b) is 0, 1,2, 3, 4, 5 or 6. Each N_(a) can independently represent anoligonucleotide sequence comprising 2-20, 2-15, or 2-10 modifiednucleotides.

Each of X, Y and Z may be the same or different from each other.

In other embodiments, i is 0 and j is 0, and the sense strand may berepresented by the formula:5′n _(p)-N _(a)-YYY-N _(a)-n _(q)3′  (Ia).

When the sense strand is represented by formula (Ia), each N_(a)independently can represent an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

In one embodiment, the antisense strand sequence of the RNAi may berepresented by formula (II):5′n _(q′)-N _(a)′-(Z′Z′Z′)_(k)-N _(b)′-Y′Y′Y′-N _(b)′-(X′X′X′)_(l)-N′_(a)-n _(p)′3′  (II)

wherein:

k and 1 are each independently 0 or 1;

p′ and q′ are each independently 0-6;

each N_(a)′ independently represents an oligonucleotide sequencecomprising 0-25 modified nucleotides, each sequence comprising at leasttwo differently modified nucleotides;

each N_(b)′ independently represents an oligonucleotide sequencecomprising 0-10 modified nucleotides;

each n_(p)′ and n_(q)′ independently represent an overhang nucleotide;

wherein N_(b)′ and Y′ do not have the same modification;

and

X′X′X′, Y′Y′Y′ and Z′Z′Z′ each independently represent one motif ofthree identical modifications on three consecutive nucleotides.

In one embodiment, the N_(a)′ and/or N_(b)′ comprise modifications ofalternating pattern.

The Y′Y′Y′ motif occurs at or near the cleavage site of the antisensestrand. For example, when the RNAi agent has a duplex region of17-23nucleotidein length, the Y′Y′Y′ motif can occur at positions 9, 10,11, 10, 11, 12; 11, 12, 13; 12, 13, 14; or 13, 14, 15 of the antisensestrand, with the count starting from the 1^(st) nucleotide, from the5′-end; or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end. Preferably, theY′Y′Y′ motif occurs at positions 11, 12, 13.

In one embodiment, Y′Y′Y′ motif is all 2′-OMe modified nucleotides.

In one embodiment, k is 1 and l is 0, or k is 0 and l is 1, or both kand l are 1.

The antisense strand can therefore be represented by the followingformulas:5′n _(q)′-N _(a)′-Z′Z′Z′—N _(b)′-Y′Y′Y′-N _(a)′-n _(p′)3′  (IIb);5′n _(q)′-N _(a)′-Y′Y′Y′-N _(b)′-X′X′X′-n _(p)′3′  (IIc); or5′n _(q)′-N _(a)′-Z′Z′Z′-N _(b)′-Y′Y′Y′-N _(b)′-X′X′X′-N _(a)′-n_(p′)3′  (IId).

When the antisense strand is represented by formula (IIb), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IIc), N_(b)′represents an oligonucleotide sequence comprising 0-10, 0-7, 0-10, 0-7,0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′ independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the antisense strand is represented as formula (IId), each N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides. Preferably, N_(b) is 0, 1, 2, 3, 4,5 or 6.

In other embodiments, k is 0 and 1 is 0 and the antisense strand may berepresented by the formula:5′n _(p)′-N _(a)′-Y′Y′Y′-N _(a)′-n _(q)′3′  (Ia).

When the antisense strand is represented as formula (IIa), each N_(a)′independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

Each of X′, Y′ and Z′ may be the same or different from each other.

Each nucleotide of the sense strand and antisense strand may beindependently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-hydroxyl, or 2′-fluoro. Forexample, each nucleotide of the sense strand and antisense strand isindependently modified with 2′-O-methyl or 2′-fluoro. Each X, Y, Z, X′,Y′ and Z′, in particular, may represent a 2′-O-methyl modification or a2′-fluoro modification.

In one embodiment, the sense strand of the RNAi agent may contain YYYmotif occurring at 9, 10 and 11 positions of the strand when the duplexregion is 21 nt, the count starting from the 1^(st) nucleotide from the5′-end, or optionally, the count starting at the 1^(st) pairednucleotide within the duplex region, from the 5′-end; and Y represents2′-F modification. The sense strand may additionally contain XXX motifor ZZZ motifs as wing modifications at the opposite end of the duplexregion; and XXX and ZZZ each independently represents a 2′-OMemodification or 2′-F modification.

In one embodiment the antisense strand may contain Y′Y′Y′ motifoccurring at positions 11, 12, 13 of the strand, the count starting fromthe 1^(st) nucleotide from the 5′-end, or optionally, the count startingat the 1^(st) paired nucleotide within the duplex region, from the5′-end; and Y′ represents 2′-O-methyl modification. The antisense strandmay additionally contain X′X′X′ motif or Z′Z′Z′ motifs as wingmodifications at the opposite end of the duplex region; and X′X′X′ andZ′Z′Z′ each independently represents a 2′-OMe modification or 2′-Fmodification.

The sense strand represented by any one of the above formulas (Ia),(Ib), (Ic), and (Id) forms a duplex with a antisense strand beingrepresented by any one of formulas (IIa), (IIb), (IIc), and (IId),respectively.

Accordingly, the RNAi agents for use in the methods of the disclosuremay comprise a sense strand and an antisense strand, each strand having14 to 30 nucleotides, the RNAi duplex represented by formula (III):sense: 5′n _(p)-N _(a)-(XXX)_(i)-N _(b)-YYY-N _(b)-(ZZZ)_(j)-N _(a)-n_(q)3′antisense: 3′n _(p)′-N _(a)′-(X′X′X′)_(k)-N _(b)′-Y′Y′Y′-N_(b)′-(Z′Z′Z′)_(l)-N _(a)′-n _(q)′5′   (III)

wherein:

j, k, and 1 are each independently 0 or 1;

p, p′, q, and q′ are each independently 0-6;

each N_(a) and N_(a)′ independently represents an oligonucleotidesequence comprising 0-25 modified nucleotides, each sequence comprisingat least two differently modified nucleotides;

each N_(b) and N_(b)′ independently represents an oligonucleotidesequence comprising 0-10 modified nucleotides;

wherein

each n_(p)′, n_(p), n_(q)′, and n_(q), each of which may or may not bepresent, independently represents an overhang nucleotide; and

XXX, YYY, ZZZ, X′X′X′, Y′Y′Y′, and Z′Z′Z′ each independently representone motif of three identical modifications on three consecutivenucleotides.

In one embodiment, i is 0 and j is 0; or i is 1 and j is 0; or i is 0and j is 1; or both i and j are 0; or both i and j are 1. In anotherembodiment, k is 0 and 1 is 0; or k is 1 and 1 is 0; k is 0 and 1 is 1;or both k and l are 0; or both k and l are 1.

Exemplary combinations of the sense strand and antisense strand forminga RNAi duplex include the formulas below:5′n _(p)-N _(a)-YYY-N _(a)-n _(q)3′3′n _(p)′-N _(a)′-Y′Y′Y′-N _(a) ′n _(q)′5′  (IIIa)5′n _(p)-N _(a)-YYY-N _(b)-ZZZ-N _(a)-n _(q)3′3′n _(p)′-N _(a)′-Y′Y′Y′-N _(b)′-Z′Z′Z′-N _(a) ′n _(q)′5′  (IIIb)5′n _(p)-N _(a)-XXX-N _(b)-YYY-N _(a)-n _(q)3′3′n _(p)′-N _(a)′-X′X′X′-N _(b)′-Y′Y′Y′—N _(a)′-n _(q)′5′  (IIIc)5′n _(p)-N _(a)-XXX-N _(b)-YYY-N _(b)′-ZZZ-N _(a)-n _(q)3′3′n _(p)′-N _(a)′-X′X′X′-N _(b)′-Y′Y′Y′—N _(b)′-Z′Z′Z′-N _(a)-n_(q)′5′  (IIId)

When the RNAi agent is represented by formula (IIIa), each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented by formula (IIIb), each N_(b)independently represents an oligonucleotide sequence comprising 1-10,1-7, 1-5 or 1-4 modified nucleotides. Each N_(a) independentlyrepresents an oligonucleotide sequence comprising 2-20, 2-15, or 2-10modified nucleotides.

When the RNAi agent is represented as formula (IIIc), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0modified nucleotides. Each N_(a)independently represents an oligonucleotide sequence comprising 2-20,2-15, or 2-10 modified nucleotides.

When the RNAi agent is represented as formula (IIId), each N_(b), N_(b)′independently represents an oligonucleotide sequence comprising 0-10,0-7, 0-10, 0-7, 0-5, 0-4, 0-2 or 0 modified nucleotides. Each N_(a),N_(a)′ independently represents an oligonucleotide sequence comprising2-20, 2-15, or 2-10 modified nucleotides. Each of N_(a), N_(a)′, N_(b)and N_(b)′ independently comprises modifications of alternating pattern.

In one embodiment, when the RNAi agent is represented by formula (IIId),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications. Inanother embodiment, when the RNAi agent is represented by formula(IIId), the N_(a) modifications are 2′-O-methyl or 2′-fluoromodifications and n_(p)′>0 and at least one n_(p)′ is linked to aneighboring nucleotide a via phosphorothioate linkage. In yet anotherembodiment, when the RNAi agent is represented by formula (IIId), theN_(a) modifications are 2′-O-methyl or 2′-fluoro modifications, n_(p)′>0and at least one n_(p)′ is linked to a neighboring nucleotide viaphosphorothioate linkage, and the sense strand is conjugated to one ormore C16 (or related) moieties attached through a bivalent or trivalentbranched linker (described below). In another embodiment, when the RNAiagent is represented by formula (IIId), the N_(a) modifications are2′-O-methyl or 2′-fluoro modifications, n_(p)′>0 and at least one n_(p)′is linked to a neighboring nucleotide via phosphorothioate linkage, thesense strand comprises at least one phosphorothioate linkage, and thesense strand is conjugated to one or more C16 (or related) moieties,optionally attached through a bivalent or trivalent branched linker.

In one embodiment, when the RNAi agent is represented by formula (IIIa),the N_(a) modifications are 2′-O-methyl or 2′-fluoro modifications,n_(p)′>0 and at least one n_(p)′ is linked to a neighboring nucleotidevia phosphorothioate linkage, the sense strand comprises at least onephosphorothioate linkage, and the sense strand is conjugated to one ormore C16 (or related) moieties attached through a bivalent or trivalentbranched linker.

In one embodiment, the RNAi agent is a multimer containing at least twoduplexes represented by formula (III), (IIIa), (IIIb), (IIIc), and(IIId), wherein the duplexes are connected by a linker. The linker canbe cleavable or non-cleavable. Optionally, the multimer furthercomprises a ligand. Each of the duplexes can target the same gene or twodifferent genes; or each of the duplexes can target same gene at twodifferent target sites.

In one embodiment, the RNAi agent is a multimer containing three, four,five, six or more duplexes represented by formula (III), (IIIa), (IIIb),(IIIc), and (IIId), wherein the duplexes are connected by a linker. Thelinker can be cleavable or non-cleavable. Optionally, the multimerfurther comprises a ligand. Each of the duplexes can target the samegene or two different genes; or each of the duplexes can target samegene at two different target sites.

In one embodiment, two RNAi agents represented by formula (III), (IIIa),(IIIb), (IIIc), and (IIId) are linked to each other at the 5′ end, andone or both of the 3′ ends and are optionally conjugated to to a ligand.Each of the agents can target the same gene or two different genes; oreach of the agents can target same gene at two different target sites.

Various publications describe multimeric RNAi agents that can be used inthe methods of the disclosure. Such publications include WO2007/091269,U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686, WO2009/014887 andWO2011/031520 the entire contents of each of which are herebyincorporated herein by reference. In certain embodiments, the RNAiagents of the disclosure may include GalNAc ligands, even if such GalNAcligands are currently projected to be of limited value for the preferredintrathecal/CNS delivery route(s) of the instant disclosure.

As described in more detail below, the RNAi agent that containsconjugations of one or more carbohydrate moieties to a RNAi agent canoptimize one or more properties of the RNAi agent. In many cases, thecarbohydrate moiety will be attached to a modified subunit of the RNAiagent. For example, the ribose sugar of one or more ribonucleotidesubunits of a dsRNA agent can be replaced with another moiety, e.g., anon-carbohydrate (preferably cyclic) carrier to which is attached acarbohydrate ligand. A ribonucleotide subunit in which the ribose sugarof the subunit has been so replaced is referred to herein as a ribosereplacement modification subunit (RRMS). A cyclic carrier may be acarbocyclic ring system, i.e., all ring atoms are carbon atoms, or aheterocyclic ring system, i.e., one or more ring atoms may be aheteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be amonocyclic ring system, or may contain two or more rings, e.g. fusedrings. The cyclic carrier may be a fully saturated ring system, or itmay contain one or more double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

The RNAi agents may be conjugated to a ligand via a carrier, wherein thecarrier can be cyclic group or acyclic group; preferably, the cyclicgroup is selected from pyrrolidinyl, pyrazolinyl, pyrazolidinyl,imidazolinyl, imidazolidinyl, piperidinyl, piperazinyl, [1,3]dioxolane,oxazolidinyl, isoxazolidinyl, morpholinyl, thiazolidinyl,isothiazolidinyl, quinoxalinyl, pyridazinonyl, tetrahydrofuryl and anddecalin; preferably, the acyclic group is selected from serinol backboneor diethanolamine backbone.

In certain specific embodiments, the RNAi agent for use in the methodsof the disclosure is an agent selected from the group of agents listedin any one of Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, and 26.These agents may further comprise a ligand.

IV. APP Knockdown to Treat APP-Associated Diseases

Certain aspects of the instant disclosure are directed to RNAiagent-mediated knockdown of APP-associated diseases or disorders, whichinclude CAA and AD, including hereditary CAA and EOFAD, as well assporadic and/or late onset AD.

Hereditary CAA (hCAA) is a vascular proteinopathy, for which the amyloidtherapeutic hypothesis is relatively straightforward and clinicallytestable. It is a devastating and rare disease, with no existingtherapy. Both biochemical and imaging biomarkers exist for clinicalvalidation of anti-APP siRNA-mediated treatment of hCAA.

One particular type of hCAA contemplated for treatment using the RNAiagents of the instant disclosure is “Dutch type” AP hCAA, which has anestimated patient population in the hundreds, primarily located in theNetherlands and Western Australia. Among APP-associated diseases, hCAAis unique in being purely vascular: in CAA, amyloid fibrils deposit inarterioles and capillaries of CNS parenchyma and leptomeninges, leadingto cognitive decline due to cerebral ischemia and microhemorrhages insubjects suffering from CAA. CAA is present in greater than 80% of allAD subjects (with 25% of AD subjects having moderate-severe CAA), andthe incidence of CAA rises with the age of a subject, at approximately50% incidence in elderly over 70 years of age.

The following are exemplary manifestations of hereditary CAA:

-   -   Amyloid-beta—Sporadic CAA, HCHWA-Dutch and Italian type EOFAD,        LOAD, Trisomy 21    -   ABri—Familial British Dementia    -   ADan—Familial Danish Dementia    -   Cystatin C—HCHWA-Icelandic type (HCHWA-Hereditary cerebral        hemorrhage with amyloidosis)    -   Gelsolin—Familial Amyloidosis-Finnish type    -   Prion protein—Prion disease    -   Transthyretin—Hereditary systemic amyloidosis

As noted above, Aβ-hCAA (aka APP-hCAA) is a rapidly progressive,dementing disease associated with intracerebral hemorrhage. Knownindications of CAA include both APP-hCAA and sporadic CAA. Possibleadditional CAA indications include: CAA associated with EOFAD (PSEN1;APP; PSEN2); CAA associated with Down syndrome; and CAA associated withlate-onset Alzheimer's disease (for which prevalence is common, as notedabove).

For APP-hCAA as an indication, the prevalence of APP-hCAA is not known;however, pure APP-hCAA is less common than EOFAD (Dutch type hCAA(involving an APP E693Q mutation) has been reported in several hundredindividuals). Typically, onset of APP-hCAA symptoms occur from age35-45; and APP-hCAA typically progresses to serious CVA within 2-5years, resulting in a peak age at death from CVA at age 55.

Sporadic CAA as an indication exhibits relatively high prevalence: it isthe common cause of lobar intracerebral hemorrhage (ICH) in the elderly.It is also a rapidly progressive disease, with 86 (36%) of 316 patientsdeveloped recurrent ICH over a mean follow up time of 5 years (Van Ettenet al. 2016 Neurology). Cumulative dementia incidence in sporadic CAAwas observed in one study to be 14% at 1 year and 73% at 5 years (Xionget al. 2017 J Cerebr Blood Flow Metab). Sporadic CAA also overlapsextensively with AD, as advanced CAA has been identified as present inapproximately 25% of AD brains; however, less than 50% of CAA casesactually meet the pathological criteria for AD.

To assess the efficacy of APP knockdown in a subject treated with a RNAiagent of the instant disclosure, it is expressly contemplated thatsoluble forms of APP, particularly including APPα and APPβ can serve ascerebrospinal fluid (CSF) biomarkers for assessing APP knockdownefficiency.

Amyloid-β production, elimination and deposition in CAA: convergingevidence indicates that the major source of Aβ is neuronal. It isgenerated by sequential cleavage of amyloid precursor protein (APP) byβ- and γ-secretases, in proportion to neuronal activity. Aβ iseliminated from the brain by four major pathways: (a) proteolyticdegradation by endopeptidases (such as neprilysin and insulin degradingenzyme (IDE)); (b) receptor mediated clearance by cells in the brainparenchyma (microglia, astrocytes and to a lesser extent neurones); (c)active transport into the blood through the blood-brain barrier (BBB);(d) elimination along the perivascular pathways by which interstitialfluid drains from the brain. Specialized carriers (e.g., ApoE) and/orreceptor transport mechanisms (eg, the low density lipoprotein receptor(LDLR) and LDLR related protein (LRP1)) are involved in all majorcellular clearance pathways. Vascular deposition is facilitated byfactors that increase the Aβ40:Aβ42 ratio (while increased Aβ42 leads tooligomerization and amyloid plaques) and impede perivascular passage. Asthe clearance mechanisms fail with age, AP is increasingly entrappedfrom the perivascular drainage pathways into the basement membranes ofcapillaries and arterioles of the brain leading to CAA. ApoE alleleshave a differential effect on different molecular and cellular processesof Aβ production, elimination and deposition in a way that they eitherincrease or decrease the risk of developing CAA (Charidimou A et al. JNeurol Neurosurg Psychiatry 2012; 83: 124-137).

Sequential cleavage of APP occurs by two pathways. The APP family ofproteins is noted as having large, biologically active, N-terminalectodomains as well as a shorter C-terminus that contains a crucialTyrosine-Glutamic Acid-Asparagine-Proline-Threonine-Tyrosine (YENPTY;SEQ ID NO: 1863) protein-sorting domain to which the adaptor proteinsX11 and Fe65 bind. The resulting Aβ peptide cleavage product startswithin the ectodomain and continues into the transmembrane region. Inone pathway, APP is cleaved by α-secretase followed by γ-secretase inperforming nonamyloidogenic processing of APP. In a second pathway,amyloidogenic processing of APP involves BACE1 cleavage followed byγ-secretase. Both processes generate soluble ectodomains (sAPPα andsAPPβ) and identical intracellular C-terminal fragments (AICD; SEQ IDNO: 1864; Thinakaran and Koo. J. Biol. Chem. 283: 29615-19; Reinhard etal. The EMBO Journal, 24: 3996-4006; Walsh et al. Biochemical SocietyTransactions, 35: 416-420; O'Brien and Wong. Annu Rev Neurosci. 34:185-204).

CAA histopathology includes morphological changes of vessel walls (asrevealed by haematoxylin-eosin staining) and Aβ deposition. Inleptomeningeal arterioles, significant structural alterations and doublebarreling have been observed (Charidimou et al. J Neurol NeurosurgPsychiatry 83: 124-137). In mild and moderate CAA, only minimalstructural changes have been detected; however, in advanced CAA,significant structural alterations have been detected, the most extremeof which is double barrelling (detachment and delamination of the outerpart of the tunica media). A similar pathological range of CAA relatedchanges in leptomeningeal arterioles have also been observed usingimmunohistochemical detection of Aβ. In mild CAA, patchy deposition ofamyloid has been observed in the wall of examined vessels. Moderate CAAhas shown more dense amyloid deposition which spans the entire vesselwall, while severe CAA has shown double balled vessels and endothelialinvolvement. Pathological findings of CAA in cortical arterioles hasrevealed progressive Aβ deposition in proportion to disease severity.Moderate CAA has shown pan-mural deposition of Aβ along with Aβdeposition in the surrounding brain parenchyma, while in severe CAA, adouble barrel vessel has been observed, although this was less common ascompared with leptomeningeal vessels (Charidimou et al.).

Pathogenesis of CAA has also been examined. Amyloid beta produced by thebrain parenchyma is normally cleared via a perivascular route. Excessiveproduction of Aβ expression of specific CAA-prone Aβ variants anddelayed drainage of Aβ has been observed to lead to amyloid depositionin the media of small arteries in the CNS. Soluble and insoluble amyloidfibrils have been identified as toxic to vascular smooth muscle and suchfibrils replace these cells, disabling vascular reactivity. Furtherdamage to the endothelium has been observed to lead to microhemorrhages,microinfarcts and tissue destruction leading to dementia. Furtherprogression has caused intracerebral hemorrhage, which has often beenobserved to be lethal. CAA has been observed to occur most frequently inthe occipital lobe, less frequently in the hippocampus, cerebellum,basal ganglia, and not normally in the deep central grey matter,subcortical white matter and brain stem (Charidimou et al.).

Many potential outcome markers have been identified for performance ofCAA human studies. In addition to symptomatic intracerebral haemorrhage,microbleeds, white matter hyperintensities (WMH) and amyloid imaginghave been associated with disease severity and progression (Greenburg etal., Lancet Neurol 13: 419-28).

Available assays can also be used to detect soluble APP levels in humanCSF samples. In particular, sAPPα and sAPPβ are soluble forms of APP andhave been identified as serving as PD (pharmacodynamic) biomarkers.Analytes have also been detected in non-human primate (NHP) CSF samples,and such assays can enable efficacy studies in NHPs. Detection ofAβ40/42/38 peptides and Total tau/P181 Tau has also been described andis being implemented in the current studies.

Imaging biomarkers are also available for CAA studies, ascerebrovascular function has been identified to reflect pathology inCAA. Imaging has been specifically used to measureblood-oxygen-level-dependent (BOLD) signal after visual stimulation (VanOpstal et al., The lancet Neurology; 16(2); 2017; Peca S et al.,Neurology. 2013; 81(19); Switzer A et al., Neurolmage Clinical; 2016).In performing BOLD fMRI in CAA subjects (assessing group blood oxygenlevel-dependent functional MRI responses for motor and visual tasks),reduced functional MRI activation has been observed for patients withCAA. In particular, BOLD fMRI activity in visual cortex has beenobserved to be correlated with higher WMH volume and higher microbleedcount (Peca et al., Neurology 2013; 81(19); Switzer et al. NeurolmageClinical 2016).

Animal models of CAA have also been described, which allow fordetermination of the effect of APP knockdown on CAA pathology andidentification of translatable biomarkers. In particular, multiplerodent models that express mutant human APP and show CAA pathology havebeen developed, including Tg-SwDI/NOS2−/−. In Tg-SwDI/NOS2−/− modelmice, increased AP levels have been identified with increased age ofmodel mice. Perivascular hyperphosphorylated tau protein has also beenassociated with capillary amyloid not only in Tg-SwDI/NOS2−/− mice butalso in human CAA-type 1 samples (Hall and Roberson. Brain Res Bull.2012; 88(1): 3-12; Attems et al., Nephrology and Applied Neurobiology,2011, 37, 75-93). A CVN mouse model of AD (APPSDI/NOS2 KO) alsoexhibited phenotypes including amyloid plaques in the hippocampus,thalamus and cortex, increased tissue inflammation and behavioraldeficits. A transgenic rat model (harboring hAPP mutations) has alsobeen developed.

Thus, APP has been identified as a target for hereditary cerebralamyloid angiopathy (CAA). Mutations in APP that have been reported tocause severe forms of CAA include A692G (Flemish), E693Q (Dutch), E693K(Italian), and D694N (Iowa). Meanwhile, mutations in APP that have beendescribed to cause early onset AD include E665D, K670N, M671L (Swedish),T714A (Iranian), T7141 (Austrian), V715M (French), V715A (German), I716V(Florida), I716T, V7171 (London), V717F, V717G and V717L. In particular,the APP E693Q (Dutch) mutation causes severe CAA with few parenchymalneurofibrillary tangles; E693Q increases amyloid beta aggregation andtoxicity; E693K (Italian) is similar but E693G (Arctic), E693A andE693delta mutations cause EOFAD with little or no CAA; and APP D694N(Iowa) causes severe CAA with typical AD pathology. In addition to thepreceding point mutations, APP duplications that result in APPoverexpression have also been identified to cause Aβ deposition.Meanwhile, no known APP mutations have been described that prevent ordelay APP-hCAA. In addition to APP mutants, Aβ CAA has also beenobserved for PSEN1 (L282V) and PSEN2 (N141I) mutations. Meanwhile, ApoEε2 (independent of AD) and ApoE ε4 (dependent on AD) have also beenreported as risk factors for CAA (Rensink A et al., Brain ResearchReviews, 43 (2) 2003).

Certain aspects of the instant disclosure are directed towards targetingof APP for knockdown in individuals having APP-hCAA. A need exists forsuch agents because there are currently no disease-modifying therapiesfor CAA. In certain embodiments, the RNAi agents of the instantdisclosure should provide approximately 60-80% knockdown of both mutantand WT APP levels throughout the CNS.

Humans with heterozygous APP mutations exist in the general populationwith pLI score of 0.3; however, no Human APP knockout has beenidentified thus far.

Pharmacological attempts to treat human CAA include the following:

-   -   Ponezumab, an amyloid beta 40 antibody was studied by Pfizer in        36 individuals with late-onset CAA Three infusions of ponezumab        or placebo over the course of 60 days were evaluated for changes        in cerebrovascular reactivity as measured by BOLD fMRI, as well        as for cerebral edema, infarcts, AP, cognitive change and other        secondary outcomes. Ponezumab showed drug-placebo differences,        but did not meet the primary endpoint.    -   BAN2401-. Amyloid beta therapeutic antibodies delivered        systemically were identified to be safe but also could cause        local cerebral edema. In a recent phase II 18-mo trial of        BAN2401 in LOAD, the incidence of SAEs was 17.6% for placebo and        15.5% for the highest dose (10 mg/kg biweekly). Amyloid Related        Imaging Abnormalities-Edema (ARIA-E) was 14.6% at the highest        dose in APOE4 carriers.

Against animal CAA models, ponezumab was noted as effective in a mousemodel of CAA with respect to lowering amyloid beta burden and vascularreactivity (Bales, 2018). Meanwhile, global APP knockout mice havefurther been noted as viable.

The following exemplary biomarker and pathological data have alsoprovided further validation for the primary role for amyloid betaprotein in pathogenesis of CAA:

-   -   Hereditary forms of “pure” CAA (i.e., lacking parenchymal plaque        amyloid) have been observed as characterized by predominant Aβ40        deposition in amyloid, as opposed to Aβ42 in parenchymal AD;    -   CAA has been observed as not a “tauopathy”, with normal levels        of T-tau and P-tau in the CSF, in contrast to elevated levels        observed in AD;    -   The inverse correlation of increasing brain amyloid burden,        measured by PiB PET, with decreasing CSF Aβ40 levels has been        identified as unique to CAA; and    -   In vitro and in vivo experimental data have provided increasing        support to a prion hypothesis in CAA, wherein A1340 containing        hereditary CAA mutations has a propensity to misfold and induce        misfolding in WT protein, so that both are present in amyloid        fibrils (akin to transthyretin (TTR)).

As disclosed in the below Examples, the instant disclosure provides anumber of mouse/rat cross reactive APP-targeting duplexes (including,e.g., AD-397177, AD397192, AD-397196, AD-397182, AD397190, AD-397265 andAD-397203), based upon screening results obtained for APP liver mRNA,when duplexes were administered at 2 mg/Kg in a single dose, as assessedat day 21 post-dosing. The instant disclosure also provides a number ofhuman/cynomolgus cross-reactive duplexes (including, e.g., AD-392911,AD-392912, AD-392703, AD-392866, AD-392927, AD-392913, AD-392843,AD-392916, AD-392714, AD-392844, AD-392926, AD-392824, AD-392704 andAD-392790), based upon screening results obtained for treatment ofprimary cynomolgus hepatocytes and human BE(2)C cells.

RNAi agent-mediated knockdown of EOFAD is also expressly contemplated.Like hCAA, EOFAD is a devastating and rare disease and—as for hCAA—acausal role of APP is well-established and phenotyping of the diseasecan be performed with greater accuracy and over a shorter duration oftime than, e.g., sporadic and/or late onset AD (optionally late onset ADwith severe CAA as a subclass of late onset AD). EOFAD is a progressive,dementing neurodegenerative disease in young adults, possessing an ageof onset before age 60 to 65 years and often before 55 years of age.

The prevalence of EOFAD has been estimated to be 41.2 per 100,000 forthe population at risk (i.e., persons aged 40-59 years), with 61% ofthose affected by EOFAD having a positive family history of EOFAD (amongthese, 13% had affected individuals in three generations). EOFADcomprises less than 3% of all AD (Bird, Genetics in Medicine, 10:231-239; Brien and Wang. Annu Rev Neu Sci, 2011, 34: 185-204; NCBI GeneReviews).

Providing human genetic validation of the APP target (OMIM 104300),certain APP mutations have been identified that cause EOFAD, includingE665D, K670N, M671L (Swedish), T714A (Iranian), T7141 (Austrian), V715M(French), V715A (German), I716V (Florida), I716T, V7171 (London), V717F,V717G and V717L, as described above. In addition, dominant amyloid betaprecursor protein mutations have also been identified that cause EOFADand CAA.

Without wishing to be bound by theory, the pathogenesis of AD isbelieved to begin in the hippocampus, a ridge of grey matter immediatelysuperior to both lateral ventricles. Degeneration of this tissue isbelieved to cause the memory loss characteristic of early disease. Whilethe mechanism of neurodegeneration at the protein level has been amatter of great debate, duplications of APP associated with EOFAD haveindicated that overexpression of APP may be sufficient to cause AD.(Haass and Selkoe. Nature Reviews Molecular Cell Biology, 8: 101-112).

In contrast to EOFAD and CAA, the pathogenic mechanisms of sporadic ADare not yet understood and the population of clinically defined sporadicAD is probably mechanistically heterogeneous.

Certain aspects of the instant disclosure are directed towards targetingof APP for knockdown in individuals having EOFAD. A need exists for suchagents because only symptom-directed treatments (of limited efficacy)exist for AD more generally and EOFAD in particular. In certainembodiments, the RNAi agents of the instant disclosure should provideapproximately 60-80% knockdown of both mutant and WT APP levelsthroughout the CNS. One further observation from human genetics thatspeaks to the likely therapeutic efficacy of an APP-targeted therapycapable of knocking down APP levels in CNS cells is that an A673Tmutation was identified that protected carriers from AD and dementia inthe general population (Jonsson et al. Nature Letter, 488.doi:doi:10.1038/nature11283). The A673T substitution is adjacent to aβ-secretase cleavage site, and has been described as resulting in a 40%reduction in amyloid beta in cell assays. Thus, a dominant negative APPpoint mutant appeared to protect families from AD, further reinforcingthat RNAi agent-mediated knockdown of APP could exert a similarprotective and/or therapeutic effect in at least certain forms of AD,including EOFAD.

Aiding initial stages of APP-targeting RNAi agent development, it hasbeen noted that APP knockout mice are viable (OMIM 104300), which isexpected to allow for viable use of mouse as a model system during leadcompound development. In contrast to mice, while humans possessingheterozygous APP mutations exist in the general population with EXACscore of 0.3, no human APP knockout has been identified to date.Biomarkers available for development of APP-targeting RNAi agentsinclude APP and MAPT peptides in CSF, which should allow for rapidassessment and useful efficacy even in a genetically homogeneouspopulation (Mo et al. (2017) Alzheimers & Dementia: Diagnosis,Assessment & Disease Monitoring, 6: 201-209).

As noted above, attempts to treat sporadic forms of AD and EOFAD have todate proven unsuccessful—for example, all trials of BACE1 (β-secretase)inhibitors (BACE1i) for treatment of sporadic AD have thus far failed(Egan et al. The New England Journal of Medicine, 378: 1691-1703; Hungand Fu. Journal of Biomedical Science, 24: 47). In such BACEi testing,there have been no completed studies in genetically-defined populations(only studies initiated). Notably, the most recent BACEli study showedthat verubecestat lowered amyloid beta levels by 60% in a populationselected based on age and clinical criteria that suggested a probablediagnosis of AD (Egan et al. The New England Journal of Medicine, 378:1691-1703; Hung and Fu. Journal of Biomedical Science, 24: 47).Meanwhile, among Aβ-directed immunotherapies, one such immunotherapydemonstrated proof-of-concept in a recent trial in sporadic AD,supporting initiation of an ongoing Phase III trial (Selkoe and Hardy.EMBO Molecular Medicine, 8: 595-608). Given its role in APP cleavage,γ-secretase has also been targeted in certain AD-directed trials.However, to date no γ-secretase inhibitor trials have been completed ina genetically-defined population; and several programs have beendiscontinued for toxicity (Selkoe and Hardy).

A need therefore exists for agents that can treat or preventAPP-associated diseases or disorders in an affected individual.

It is expressly contemplated that all APP-associated diseases ordisorders can ultimately be targeted using the RNAi agents of theinstant disclosure—specifically, targeting of sporadic CAA and sporadicand/or late onset AD is also contemplated for the RNAi agents of theinstant disclosure, even in view of the diagnostic/phenotyping issuespresently confronted for these particular APP-associated diseases (it isfurther contemplated that diagnostics for these diseases will alsocontinue to improve).

V. RNAi Agents Conjugated to Ligands

Another modification of the RNA of a RNAi agent of the disclosureinvolves chemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the RNAi. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., (1989) Proc.Natl. Acid. Sci. USA, 86: 6553-6556), cholic acid (Manoharan et al.,(1994) Biorg. Med. Chem. Let., 4:1053-1060), a thioether, e.g.,beryl-S-tritylthiol (Manoharan et al., (1992) Ann. N.Y. Acad. Sci.,660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let.,3:2765-2770), a thiocholesterol (Oberhauser et al., (1992) Nucl. AcidsRes., 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., (1991) EMBO J, 10:1111-1118; Kabanovet al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993)Biochimie, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654; Shea et al.,(1990) Nucl. Acids Res., 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., (1995) Nucleosides & Nucleotides,14:969-973), or adamantane acetic acid (Manoharan et al., (1995)Tetrahedron Lett., 36:3651-3654), a palmityl moiety (Mishra et al.,(1995) Biochim. Biophys. Acta, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., (1996) J.Pharmacol. Exp. Ther., 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of a RNAi agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine orhyaluronic acid); or a lipid. The ligand can also be a recombinant orsynthetic molecule, such as a synthetic polymer, e.g., a syntheticpolyamino acid. Examples of polyamino acids include polyamino acid is apolylysine (PLL), poly L-aspartic acid, poly L-glutamic acid,styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied)copolymer, divinyl ether-maleic anhydride copolymer,N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol(PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllicacid), N-isopropylacrylamide polymers, or polyphosphazine. Example ofpolyamines include: polyethylenimine, polylysine (PLL), spermine,spermidine, polyamine, pseudopeptide-polyamine, peptidomimeticpolyamine, dendrimer polyamine, arginine, amidine, protamine, cationiclipid, cationic porphyrin, quaternary salt of a polyamine, or an alphahelical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, vitamin A, biotin, or an RGDpeptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, 03-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a CNScell. Ligands can also include hormones and hormone receptors. They canalso include non-peptidic species, such as lipids, lectins,carbohydrates, vitamins, cofactors, multivalent lactose, multivalentgalactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalentmannose, or multivalent fucose.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the RNAi agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In some embodiments, a ligand attached to a RNAi agent as describedherein acts as a pharmacokinetic modulator (PK modulator). PK modulatorsinclude lipophiles, bile acids, steroids, phospholipid analogues,peptides, protein binding agents, PEG, vitamins etc. Exemplary PKmodulators include, but are not limited to, cholesterol, fatty acids,cholic acid, lithocholic acid, dialkylglycerides, diacylglyceride,phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotinetc. Oligonucleotides that comprise a number of phosphorothioatelinkages are also known to bind to serum protein, thus shortoligonucleotides, e.g., oligonucleotides of about 5 bases, 10 bases, 15bases or 20 bases, comprising multiple of phosphorothioate linkages inthe backbone are also amenable to the present disclosure as ligands(e.g. as PK modulating ligands). In addition, aptamers that bind serumcomponents (e.g. serum proteins) are also suitable for use as PKmodulating ligands in the embodiments described herein.

Ligand-conjugated oligonucleotides of the disclosure may be synthesizedby the use of an oligonucleotide that bears a pendant reactivefunctionality, such as that derived from the attachment of a linkingmolecule onto the oligonucleotide (described below). This reactiveoligonucleotide may be reacted directly with commercially-availableligands, ligands that are synthesized bearing any of a variety ofprotecting groups, or ligands that have a linking moiety attachedthereto.

The oligonucleotides used in the conjugates of the present disclosuremay be conveniently and routinely made through the well-known techniqueof solid-phase synthesis. Equipment for such synthesis is sold byseveral vendors including, for example, Applied Biosystems (Foster City,Calif.). Any other means for such synthesis known in the art mayadditionally or alternatively be employed. It is also known to usesimilar techniques to prepare other oligonucleotides, such as thephosphorothioates and alkylated derivatives.

In the ligand-conjugated oligonucleotides and ligand-molecule bearingsequence-specific linked nucleosides of the present disclosure, theoligonucleotides and oligonucleosides may be assembled on a suitable DNAsynthesizer utilizing standard nucleotide or nucleoside precursors, ornucleotide or nucleoside conjugate precursors that already bear thelinking moiety, ligand-nucleotide or nucleoside-conjugate precursorsthat already bear the ligand molecule, or non-nucleoside ligand-bearingbuilding blocks.

When using nucleotide-conjugate precursors that already bear a linkingmoiety, the synthesis of the sequence-specific linked nucleosides istypically completed, and the ligand molecule is then reacted with thelinking moiety to form the ligand-conjugated oligonucleotide. In someembodiments, the oligonucleotides or linked nucleosides of the presentdisclosure are synthesized by an automated synthesizer usingphosphoramidites derived from ligand-nucleoside conjugates in additionto the standard phosphoramidites and non-standard phosphoramidites thatare commercially available and routinely used in oligonucleotidesynthesis.

A. Lipophilic Moieties

In certain embodiments, the lipophilic moiety is an aliphatic, cyclicsuch as alicyclic, or polycyclic such as polyalicyclic compound, such asa steroid (e.g., sterol) or a linear or branched aliphatic hydrocarbon.The lipophilic moiety may generally comprises a hydrocarbon chain, whichmay be cyclic or acyclic. The hydrocarbon chain may comprise varioussubstituents and/or one or more heteroatoms, such as an oxygen ornitrogen atom. Such lipophilic aliphatic moieties include, withoutlimitation, saturated or unsaturated C₄-C₃₀ hydrocarbon (e.g., C₆-C₁₈hydrocarbon), saturated or unsaturated fatty acids, waxes (e.g.,monohydric alcohol esters of fatty acids and fatty diamides), terpenes(e.g., C₁₀ terpenes, C₁₅ sesquiterpenes, C₂₀ diterpenes, C₃₀triterpenes, and C₄₀ tetraterpenes), and other polyalicyclichydrocarbons. For instance, the lipophilic moiety may contain a C₄-C₃₀hydrocarbon chain (e.g., C₄-C₃₀ alkyl or alkenyl). In some embodimentthe lipophilic moiety contains a saturated or unsaturated C₆-C₁₈hydrocarbon chain (e.g., a linear C₆-C₁₈ alkyl or alkenyl). In oneembodiment, the lipophilic moiety contains a saturated or unsaturatedC₁₆ hydrocarbon chain (e.g., a linear C₁₆ alkyl or alkenyl).

The lipophilic moiety may be attached to the RNAi agent by any methodknown in the art, including via a functional grouping already present inthe lipophilic moiety or introduced into the RNAi agent, such as ahydroxy group (e.g., —CO—CH₂—OH). The functional groups already presentin the lipophilic moiety or introduced into the RNAi agent include, butare not limited to, hydroxyl, amine, carboxylic acid, sulfonate,phosphate, thiol, azide, and alkyne.

Conjugation of the RNAi agent and the lipophilic moiety may occur, forexample, through formation of an ether or a carboxylic or carbamoylester linkage between the hydroxy and an alkyl group R—, an alkanoylgroup RCO— or a substituted carbamoyl group RNHCO—. The alkyl group Rmay be cyclic (e.g., cyclohexyl) or acyclic (e.g., straight-chained orbranched; and saturated or unsaturated). Alkyl group R may be a butyl,pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl, tridecyl,tetradecyl, pentadecyl, hexadecyl, heptadecyl or octadecyl group, or thelike.

In some embodiments, the lipophilic moiety is conjugated to thedouble-stranded RNAi agent via a linker a linker containing an ether,thioether, urea, carbonate, amine, amide, maleimide-thioether,disulfide, phosphodiester, sulfonamide linkage, a product of a clickreaction (e.g., a triazole from the azide-alkyne cycloaddition), orcarbamate.

In another embodiment, the lipophilic moiety is a steroid, such assterol. Steroids are polycyclic compounds containing aperhydro-1,2-cyclopentanophenanthrene ring system. Steroids include,without limitation, bile acids (e.g., cholic acid, deoxycholic acid anddehydrocholic acid), cortisone, digoxigenin, testosterone, cholesterol,and cationic steroids, such as cortisone. A “cholesterol derivative”refers to a compound derived from cholesterol, for example bysubstitution, addition or removal of substituents.

In another embodiment, the lipophilic moiety is an aromatic moiety. Inthis context, the term “aromatic” refers broadly to mono- andpolyaromatic hydrocarbons. Aromatic groups include, without limitation,C₆-C₁₄ aryl moieties comprising one to three aromatic rings, which maybe optionally substituted; “aralkyl” or “arylalkyl” groups comprising anaryl group covalently linked to an alkyl group, either of which mayindependently be optionally substituted or unsubstituted; and“heteroaryl” groups. As used herein, the term “heteroaryl” refers togroups having 5 to 14 ring atoms, preferably 5, 6, 9, or 10 ring atoms;having 6, 10, or 14n electrons shared in a cyclic array, and having, inaddition to carbon atoms, between one and about three heteroatomsselected from the group consisting of nitrogen (N), oxygen (O), andsulfur (S).

As employed herein, a “substituted” alkyl, cycloalkyl, aryl, heteroaryl,or heterocyclic group is one having between one and about four,preferably between one and about three, more preferably one or two,non-hydrogen substituents. Suitable substituents include, withoutlimitation, halo, hydroxy, nitro, haloalkyl, alkyl, alkaryl, aryl,aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbamoyl,arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl,alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido,aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups.

In some embodiments, the lipophilic moiety is an aralkyl group, e.g., a2-arylpropanoyl moiety. The structural features of the aralkyl group areselected so that the lipophilic moiety will bind to at least one proteinin vivo. In certain embodiments, the structural features of the aralkylgroup are selected so that the lipophilic moiety binds to serum,vascular, or cellular proteins. In certain embodiments, the structuralfeatures of the aralkyl group promote binding to albumin, animmunoglobulin, a lipoprotein, α-2-macroglubulin, or α-1-glycoprotein.

In certain embodiments, the ligand is naproxen or a structuralderivative of naproxen. Procedures for the synthesis of naproxen can befound in U.S. Pat. Nos. 3,904,682 and 4,009,197, which are hereyincorporated by reference in their entirety. Naproxen has the chemicalname (S)-6-Methoxy-α-methyl-2-naphthaleneacetic acid and the structureis

In certain embodiments, the ligand is ibuprofen or a structuralderivative of ibuprofen. Procedures for the synthesis of ibuprofen canbe found in U.S. Pat. No. 3,228,831, which are herey incorporated byreference in their entirety. The structure of ibuprofen is

Additional exemplary aralkyl groups are illustrated in U.S. Pat. No.7,626,014, which is incorporated herein by reference in its entirety.

In another embodiment, suitable lipophilic moieties include lipid,cholesterol, retinoic acid, cholic acid, adamantane acetic acid,1-pyrene butyric acid, dihydrotestosterone,1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyanol, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,ibuprofen, naproxen, dimethoxytrityl, or phenoxazine.

In certain embodiments, more than one lipophilic moieties can beincorporated into the double-strand RNAi agent, particularly when thelipophilic moiety has a low lipophilicity or hydrophobicity. In oneembodiment, two or more lipophilic moieties are incorporated into thesame strand of the double-strand RNAi agent. In one embodiment, eachstrand of the double-strand RNAi agent has one or more lipophilicmoieties incorporated. In one embodiment, two or more lipophilicmoieties are incorporated into the same position (i.e., the samenucleobase, same sugar moiety, or same internucleosidic linkage) of thedouble-strand RNAi agent. This can be achieved by, e.g., conjugating thetwo or more lipophilic moieties via a carrier, and/or conjugating thetwo or more lipophilic moieties via a branched linker, and/orconjugating the two or more lipophilic moieties via one or more linkers,with one or more linkers linking the lipophilic moieties consecutively.

The lipophilic moiety may be conjugated to the RNAi agent via a directattachment to the ribosugar of the RNAi agent. Alternatively, thelipophilic moiety may be conjugated to the double-strand RNAi agent viaa linker or a carrier.

In certain embodiments, the lipophilic moiety may be conjugated to theRNAi agent via one or more linkers (tethers).

In one embodiment, the lipophilic moiety is conjugated to thedouble-stranded RNAi agent via a linker containing an ether, thioether,urea, carbonate, amine, amide, maleimide-thioether, disulfide,phosphodiester, sulfonamide linkage, a product of a click reaction(e.g., a triazole from the azide-alkyne cycloaddition), or carbamate.

Exemplary linkers, tethers, carriers, nucleic acid modifications,conjugates, ligands and other moieties useful for achieving centralnervous system-directed delivery of the APP-targeting RNAi agents of theinstant disclosure are described in additional detail, e.g., in U.S.Application Nos. 62/668,072, 62/738,747 and/or 62/773,082, the entirecontents of which are incorporated herein by this reference.

B. Lipid Conujugates

In one embodiment, the ligand or conjugate is a lipid or lipid-basedmolecule. Such a lipid or lipid-based molecule preferably binds a serumprotein, e.g., human serum albumin (HSA). An HSA binding ligand allowsfor vascular distribution of the conjugate to a target tissue, e.g., anon-kidney target tissue of the body. In certain embodiments, the targettissue can be the CNS, including glial cells of the brain. Othermolecules that can bind HSA can also be used as ligands. For example,neproxin or aspirin can be used. A lipid or lipid-based ligand can (a)increase resistance to degradation of the conjugate, (b) increasetargeting or transport into a target cell or cell membrane, and/or (c)can be used to adjust binding to a serum protein, e.g., HSA.

A lipid based ligand can be used to inhibit, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

Optionally, the lipid based ligand binds HSA. Preferably, it binds HSAwith a sufficient affinity such that the conjugate will be preferablydistributed to a non-kidney tissue. However, it is preferred that theaffinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bytarget cells such as brain cells. Also included are HSA and low densitylipoprotein (LDL).

C. Cell Permeation Agents

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to RNAi agentscan affect pharmacokinetic distribution of the RNAi agent, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO: 29). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO: 30) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO: 31) andthe Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 32)have been found to be capable of functioning as delivery peptides. Apeptide or peptidomimetic can be encoded by a random sequence of DNA,such as a peptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Examples of a peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit for cell targeting purposesis an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide for use in the compositions and methods of the disclosuremay be linear or cyclic, and may be modified, e.g., glyciosylated ormethylated, to facilitate targeting to a specific tissue(s).RGD-containing peptides and peptidiomimemtics may include D-amino acids,as well as synthetic RGD mimics. In addition to RGD, one can use othermoieties that target the integrin ligand. Preferred conjugates of thisligand target PECAM-1 or VEGF.

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

D. Carbohydrate Conjugates and Ligands

In some embodiments of the compositions and methods of the disclosure,an RNAi agent oligonucleotide further comprises a carbohydrate. Thecarbohydrate conjugated RNAi agents are advantageous for the in vivodelivery of nucleic acids, as well as compositions suitable for in vivotherapeutic use, as described herein. As used herein, “carbohydrate”refers to a compound which is either a carbohydrate per se made up ofone or more monosaccharide units having at least 6 carbon atoms (whichcan be linear, branched or cyclic) with an oxygen, nitrogen or sulfuratom bonded to each carbon atom; or a compound having as a part thereofa carbohydrate moiety made up of one or more monosaccharide units eachhaving at least six carbon atoms (which can be linear, branched orcyclic), with an oxygen, nitrogen or sulfur atom bonded to each carbonatom. Representative carbohydrates include the sugars (mono-, di-, tri-and oligosaccharides containing from about 4, 5, 6, 7, 8, or 9monosaccharide units), and polysaccharides such as starches, glycogen,cellulose and polysaccharide gums. Specific monosaccharides include C5and above (e.g., C5, C6, C7, or C8) sugars; di- and trisaccharidesinclude sugars having two or three monosaccharide units (e.g., C5, C6,C7, or C8).

In one embodiment, a carbohydrate conjugate for use in the compositionsand methods of the disclosure is a monosaccharide.

In certain embodiments, the compositions and methods of the disclosureinclude a C16 ligand. In exemplary embodiments, the C16 ligand of thedisclosure has the following structure (exemplified here below for auracil base, yet attachment of the C16 ligand is contemplated for anucleotide presenting any base (C, G, A, etc.) and/or possessing anyother modification as presented herein, provided that 2′ ribo attachmentis preserved) and is attached at the 2′ position of the ribo within aresidue that is so modified:

As shown above, a C16 ligand-modified residue presents a straight chainalkyl at the 2′-ribo position of an exemplary residue (here, a Uracil)that is so modified.

In some embodiments, a carbohydrate conjugate of a RNAi agent of theinstant disclosure further comprises one or more additional ligands asdescribed above, such as, but not limited to, a PK modulator and/or acell permeation peptide.

Additional carbohydrate conjugates (and linkers) suitable for use in thepresent disclosure include those described in PCT Publication Nos. WO2014/179620 and WO 2014/179627, the entire contents of each of which areincorporated herein by reference.

In certain embodiments, the compositions and methods of the disclosureinclude a vinyl phosponate (VP) modification of an RNAi agent asdescribed herein. In exemplary embodiments, a vinyl phosphonate of thedisclosure has the following structure:

A vinyl phosponate of the instant disclosure may be attached to eitherthe antisense or the sense strand of a dsRNA of the disclosure. Incertain preferred embodiments, a vinyl phosphonate of the instantdisclosure is attached to the antisense strand of a dsRNA, optionally atthe 5′ end of the antisense strand of the dsRNA.

Vinyl phosphate modifications are also contemplated for the compositionsand methods of the instant disclosure. An exemplary vinyl phosphatestructure is:

E. Thermally Destabilizing Modifications

In certain embodiments, a dsRNA molecule can be optimized for RNAinterference by incorporating thermally destabilizing modifications inthe seed region of the antisense strand (i.e., at positions 2-9 of the5′-end of the antisense strand) to reduce or inhibit off-target genesilencing. It has been discovered that dsRNAs with an antisense strandcomprising at least one thermally destabilizing modification of theduplex within the first 9 nucleotide positions, counting from the 5′end, of the antisense strand have reduced off-target gene silencingactivity. Accordingly, in some embodiments, the antisense strandcomprises at least one (e.g., one, two, three, four, five or more)thermally destabilizing modification of the duplex within the first 9nucleotide positions of the 5′ region of the antisense strand. In someembodiments, one or more thermally destabilizing modification(s) of theduplex is/are located in positions 2-9, or preferably positions 4-8,from the 5′-end of the antisense strand. In some further embodiments,the thermally destabilizing modification(s) of the duplex is/are locatedat position 6, 7 or 8 from the 5′-end of the antisense strand. In stillsome further embodiments, the thermally destabilizing modification ofthe duplex is located at position 7 from the 5′-end of the antisensestrand. The term “thermally destabilizing modification(s)” includesmodification(s) that would result with a dsRNA with a lower overallmelting temperature (Tm) (preferably a Tm with one, two, three or fourdegrees lower than the Tm of the dsRNA without having suchmodification(s). In some embodiments, the thermally destabilizingmodification of the duplex is located at position 2, 3, 4, 5 or 9 fromthe 5′-end of the antisense strand.

The thermally destabilizing modifications can include, but are notlimited to, abasic modification; mismatch with the opposing nucleotidein the opposing strand; and sugar modification such as 2′-deoxymodification or acyclic nucleotide, e.g., unlocked nucleic acids (UNA)or glycol nucleic acid (GNA).

Exemplified abasic modifications include, but are not limited to thefollowing:

Wherein R═H, Me, Et or OMe; R′═H, Me, Et or OMe; R″═H, Me, Et or OMe

wherein B is a modified or unmodified nucleobase.

Exemplified sugar modifications include, but are not limited to thefollowing:

wherein B is a modified or unmodified nucleobase.

In some embodiments the thermally destabilizing modification of theduplex is selected from the group consisting of:

wherein B is a modified or unmodified nucleobase and the asterisk oneach structure represents either R, S or racemic.

The term “acyclic nucleotide” refers to any nucleotide having an acyclicribose sugar, for example, where any of bonds between the ribose carbons(e.g., C1′-C2′, C2′-C3′, C3′-C4′, C4′-O4′, or C1′-O4′) is absent and/orat least one of ribose carbons or oxygen (e.g., C1′, C2′, C3′, C4′ orO4′) are independently or in combination absent from the nucleotide. Insome embodiments, acyclic nucleotide is

wherein B is a modified or unmodified nucleobase, R¹ and R²independently are H, halogen, OR₃, or alkyl; and R₃ is H, alkyl,cycloalkyl, aryl, aralkyl, heteroaryl or sugar). The term “UNA” refersto unlocked acyclic nucleic acid, wherein any of the bonds of the sugarhas been removed, forming an unlocked “sugar” residue. In one example,UNA also encompasses monomers with bonds between C1′-C4′ being removed(i.e. the covalent carbon-oxygen-carbon bond between the C1′ and C4′carbons). In another example, the C2′-C3′ bond (i.e. the covalentcarbon-carbon bond between the C2′ and C3′ carbons) of the sugar isremoved (see Mikhailov et. al., Tetrahedron Letters, 26 (17): 2059(1985); and Fluiter et al., Mol. Biosyst., 10: 1039 (2009), which arehereby incorporated by reference in their entirety). The acyclicderivative provides greater backbone flexibility without affecting theWatson-Crick pairings. The acyclic nucleotide can be linked via 2′-5′ or3′-5′ linkage.

The term ‘GNA’ refers to glycol nucleic acid which is a polymer similarto DNA or RNA but differing in the composition of its “backbone” in thatis composed of repeating glycerol units linked by phosphodiester bonds:

The thermally destabilizing modification of the duplex can be mismatches(i.e., noncomplementary base pairs) between the thermally destabilizingnucleotide and the opposing nucleotide in the opposite strand within thedsRNA duplex. Exemplary mismatch base pairs include G:G, G:A, G:U, G:T,A:A, A:C, C:C, C:U, C:T, U:U, T:T, U:T, or a combination thereof. Othermismatch base pairings known in the art are also amenable to the presentinvention. A mismatch can occur between nucleotides that are eithernaturally occurring nucleotides or modified nucleotides, i.e., themismatch base pairing can occur between the nucleobases from respectivenucleotides independent of the modifications on the ribose sugars of thenucleotides. In certain embodiments, the dsRNA molecule contains atleast one nucleobase in the mismatch pairing that is a 2′-deoxynucleobase; e.g., the 2′-deoxy nucleobase is in the sense strand.

In some embodiments, the thermally destabilizing modification of theduplex in the seed region of the antisense strand includes nucleotideswith impaired W—C H-bonding to complementary base on the target mRNA,such as:

More examples of abasic nucleotide, acyclic nucleotide modifications(including UNA and GNA), and mismatch modifications have been describedin detail in WO 2011/133876, which is herein incorporated by referencein its entirety.

The thermally destabilizing modifications may also include universalbase with reduced or abolished capability to form hydrogen bonds withthe opposing bases, and phosphate modifications.

In some embodiments, the thermally destabilizing modification of theduplex includes nucleotides with non-canonical bases such as, but notlimited to, nucleobase modifications with impaired or completelyabolished capability to form hydrogen bonds with bases in the oppositestrand. These nucleobase modifications have been evaluated fordestabilization of the central region of the dsRNA duplex as describedin WO 2010/0011895, which is herein incorporated by reference in itsentirety. Exemplary nucleobase modifications are:

In some embodiments, the thermally destabilizing modification of theduplex in the seed region of the antisense strand includes one or moreα-nucleotide complementary to the base on the target mRNA, such as:

wherein R is H, OH, OCH₃, F, NH₂, NHMe, NMe₂ or O-alkyl.

Exemplary phosphate modifications known to decrease the thermalstability of dsRNA duplexes compared to natural phosphodiester linkagesare:

The alkyl for the R group can be a C₁-C₆alkyl. Specific alkyls for the Rgroup include, but are not limited to methyl, ethyl, propyl, isopropyl,butyl, pentyl and hexyl. As the skilled artisan will recognize, in viewof the functional role of nucleobases is defining specificity of a RNAiagent of the disclosure, while nucleobase modifications can be performedin the various manners as described herein, e.g., to introducedestabilizing modifications into a RNAi agent of the disclosure, e.g.,for purpose of enhancing on-target effect relative to off-target effect,the range of modifications available and, in general, present upon RNAiagents of the disclosure tends to be much greater for non-nucleobasemodifications, e.g., modifications to sugar groups and/or phosphatebackbones of polyribonucleotides. Such modifications are described ingreater detail in other sections of the instant disclosure and areexpressly contemplated for RNAi agents of the disclosure, eitherpossessing native nucleobases or modified nucleobases as described aboveand/or elsewhere herein.

In addition to the antisense strand comprising a thermally destabilizingmodification, the dsRNA can also comprise one or more stabilizingmodifications. For example, the dsRNA can comprise at least two (e.g.,two, three, four, five, six, seven, eight, nine, ten or more)stabilizing modifications. Without limitations, the stabilizingmodifications all can be present in one strand. In some embodiments,both the sense and the antisense strands comprise at least twostabilizing modifications. The stabilizing modification can occur on anynucleotide of the sense strand or antisense strand. For instance, thestabilizing modification can occur on every nucleotide on the sensestrand and/or antisense strand; each stabilizing modification can occurin an alternating pattern on the sense strand or antisense strand; orthe sense strand or antisense strand comprises both stabilizingmodification in an alternating pattern. The alternating pattern of thestabilizing modifications on the sense strand may be the same ordifferent from the antisense strand, and the alternating pattern of thestabilizing modifications on the sense strand can have a shift relativeto the alternating pattern of the stabilizing modifications on theantisense strand.

In some embodiments, the antisense strand comprises at least two (e.g.,two, three, four, five, six, seven, eight, nine, ten or more)stabilizing modifications. Without limitations, a stabilizingmodification in the antisense strand can be present at any positions. Insome embodiments, the antisense comprises stabilizing modifications atpositions 2, 6, 8, 9, 14 and 16 from the 5′-end. In some otherembodiments, the antisense comprises stabilizing modifications atpositions 2, 6, 14 and 16 from the 5′-end. In still some otherembodiments, the antisense comprises stabilizing modifications atpositions 2, 14 and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least onestabilizing modification adjacent to the destabilizing modification. Forexample, the stabilizing modification can be the nucleotide at the5′-end or the 3′-end of the destabilizing modification, i.e., atposition −1 or +1 from the position of the destabilizing modification.In some embodiments, the antisense strand comprises a stabilizingmodification at each of the 5′-end and the 3′-end of the destabilizingmodification, i.e., positions −1 and +1 from the position of thedestabilizing modification.

In some embodiments, the antisense strand comprises at least twostabilizing modifications at the 3′-end of the destabilizingmodification, i.e., at positions +1 and +2 from the position of thedestabilizing modification.

In some embodiments, the sense strand comprises at least two (e.g., two,three, four, five, six, seven, eight, nine, ten or more) stabilizingmodifications. Without limitations, a stabilizing modification in thesense strand can be present at any positions. In some embodiments, thesense strand comprises stabilizing modifications at positions 7, 10 and11 from the 5′-end. In some other embodiments, the sense strandcomprises stabilizing modifications at positions 7, 9, 10 and 11 fromthe 5′-end. In some embodiments, the sense strand comprises stabilizingmodifications at positions opposite or complimentary to positions 11, 12and 15 of the antisense strand, counting from the 5′-end of theantisense strand. In some other embodiments, the sense strand comprisesstabilizing modifications at positions opposite or complimentary topositions 11, 12, 13 and 15 of the antisense strand, counting from the5′-end of the antisense strand. In some embodiments, the sense strandcomprises a block of two, three or four stabilizing modifications.

In some embodiments, the sense strand does not comprise a stabilizingmodification in position opposite or complimentary to the thermallydestabilizing modification of the duplex in the antisense strand.

Exemplary thermally stabilizing modifications include, but are notlimited to 2′-fluoro modifications. Other thermally stabilizingmodifications include, but are not limited to LNA.

In some embodiments, the dsRNA of the disclosure comprises at least four(e.g., four, five, six, seven, eight, nine, ten or more) 2′-fluoronucleotides. Without limitations, the 2′-fluoro nucleotides all can bepresent in one strand. In some embodiments, both the sense and theantisense strands comprise at least two 2′-fluoro nucleotides. The2′-fluoro modification can occur on any nucleotide of the sense strandor antisense strand. For instance, the 2′-fluoro modification can occuron every nucleotide on the sense strand and/or antisense strand; each2′-fluoro modification can occur in an alternating pattern on the sensestrand or antisense strand; or the sense strand or antisense strandcomprises both 2′-fluoro modifications in an alternating pattern. Thealternating pattern of the 2′-fluoro modifications on the sense strandmay be the same or different from the antisense strand, and thealternating pattern of the 2′-fluoro modifications on the sense strandcan have a shift relative to the alternating pattern of the 2′-fluoromodifications on the antisense strand.

In some embodiments, the antisense strand comprises at least two (e.g.,two, three, four, five, six, seven, eight, nine, ten or more) 2′-fluoronucleotides. Without limitations, a 2′-fluoro modification in theantisense strand can be present at any positions. In some embodiments,the antisense comprises 2′-fluoro nucleotides at positions 2, 6, 8, 9,14 and 16 from the 5′-end. In some other embodiments, the antisensecomprises 2′-fluoro nucleotides at positions 2, 6, 14 and 16 from the5′-end. In still some other embodiments, the antisense comprises2′-fluoro nucleotides at positions 2, 14 and 16 from the 5′-end.

In some embodiments, the antisense strand comprises at least one2′-fluoro nucleotide adjacent to the destabilizing modification. Forexample, the 2′-fluoro nucleotide can be the nucleotide at the 5′-end orthe 3′-end of the destabilizing modification, i.e., at position −1 or +1from the position of the destabilizing modification. In someembodiments, the antisense strand comprises a 2′-fluoro nucleotide ateach of the 5′-end and the 3′-end of the destabilizing modification,i.e., positions −1 and +1 from the position of the destabilizingmodification.

In some embodiments, the antisense strand comprises at least two2′-fluoro nucleotides at the 3′-end of the destabilizing modification,i.e., at positions +1 and +2 from the position of the destabilizingmodification.

In some embodiments, the sense strand comprises at least two (e.g., two,three, four, five, six, seven, eight, nine, ten or more) 2′-fluoronucleotides. Without limitations, a 2′-fluoro modification in the sensestrand can be present at any positions. In some embodiments, theantisense comprises 2′-fluoro nucleotides at positions 7, 10 and 11 fromthe 5′-end. In some other embodiments, the sense strand comprises2′-fluoro nucleotides at positions 7, 9, 10 and 11 from the 5′-end. Insome embodiments, the sense strand comprises 2′-fluoro nucleotides atpositions opposite or complimentary to positions 11, 12 and 15 of theantisense strand, counting from the 5′-end of the antisense strand. Insome other embodiments, the sense strand comprises 2′-fluoro nucleotidesat positions opposite or complimentary to positions 11, 12, 13 and 15 ofthe antisense strand, counting from the 5′-end of the antisense strand.In some embodiments, the sense strand comprises a block of two, three orfour 2′-fluoro nucleotides.

In some embodiments, the sense strand does not comprise a 2′-fluoronucleotide in position opposite or complimentary to the thermallydestabilizing modification of the duplex in the antisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprises a 21nucleotides (nt) sense strand and a 23 nucleotides (nt) antisense,wherein the antisense strand contains at least one thermallydestabilizing nucleotide, where the at least one thermally destabilizingnucleotide occurs in the seed region of the antisense strand (i.e., atposition 2-9 of the 5′-end of the antisense strand), wherein one end ofthe dsRNA is blunt, while the other end is comprises a 2 nt overhang,and wherein the dsRNA optionally further has at least one (e.g., one,two, three, four, five, six or all seven) of the followingcharacteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoromodifications; (ii) the antisense comprises 1, 2, 3, 4 or 5phosphorothioate internucleotide linkages; (iii) the sense strand isconjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 52′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5phosphorothioate internucleotide linkages; (vi) the dsRNA comprises atleast four 2′-fluoro modifications; and (vii) the dsRNA comprises ablunt end at 5′-end of the antisense strand. Preferably, the 2 ntoverhang is at the 3′-end of the antisense.

In some embodiments, the dsRNA molecule of the disclosure comprising asense and antisense strands, wherein: the sense strand is 25-30nucleotide residues in length, wherein starting from the 5′ terminalnucleotide (position 1), positions 1 to 23 of said sense strand compriseat least 8 ribonucleotides; antisense strand is 36-66 nucleotideresidues in length and, starting from the 3′ terminal nucleotide, atleast 8 ribonucleotides in the positions paired with positions 1-23 ofsense strand to form a duplex; wherein at least the 3 ‘ terminalnucleotide of antisense strand is unpaired with sense strand, and up to6 consecutive 3’ terminal nucleotides are unpaired with sense strand,thereby forming a 3′ single stranded overhang of 1-6 nucleotides;wherein the 5′ terminus of antisense strand comprises from 10-30consecutive nucleotides which are unpaired with sense strand, therebyforming a 10-30 nucleotide single stranded 5′ overhang; wherein at leastthe sense strand 5′ terminal and 3′ terminal nucleotides are base pairedwith nucleotides of antisense strand when sense and antisense strandsare aligned for maximum complementarity, thereby forming a substantiallyduplexed region between sense and antisense strands; and antisensestrand is sufficiently complementary to a target RNA along at least 19ribonucleotides of antisense strand length to reduce target geneexpression when said double stranded nucleic acid is introduced into amammalian cell; and wherein the antisense strand contains at least onethermally destabilizing nucleotide, where at least one thermallydestabilizing nucleotide is in the seed region of the antisense strand(i.e. at position 2-9 of the 5′-end of the antisense strand). Forexample, the thermally destabilizing nucleotide occurs between positionsopposite or complimentary to positions 14-17 of the 5′-end of the sensestrand, and wherein the dsRNA optionally further has at least one (e.g.,one, two, three, four, five, six or all seven) of the followingcharacteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoromodifications; (ii) the antisense comprises 1, 2, 3, 4 or 5phosphorothioate internucleotide linkages; (iii) the sense strand isconjugated with a ligand; (iv) the sense strand comprises 2, 3, 4 or 52′-fluoro modifications; (v) the sense strand comprises 1, 2, 3, 4 or 5phosphorothioate internucleotide linkages; and (vi) the dsRNA comprisesat least four 2′-fluoro modifications; and (vii) the dsRNA comprises aduplex region of 12-30 nucleotide pairs in length.

In some embodiments, the dsRNA molecule of the disclosure comprises asense and antisense strands, wherein said dsRNA molecule comprises asense strand having a length which is at least 25 and at most 29nucleotides and an antisense strand having a length which is at most 30nucleotides with the sense strand comprises a modified nucleotide thatis susceptible to enzymatic degradation at position 11 from the 5′end,wherein the 3′ end of said sense strand and the 5′ end of said antisensestrand form a blunt end and said antisense strand is 1-4 nucleotideslonger at its 3′ end than the sense strand, wherein the duplex regionwhich is at least 25 nucleotides in length, and said antisense strand issufficiently complementary to a target mRNA along at least 19 nt of saidantisense strand length to reduce target gene expression when said dsRNAmolecule is introduced into a mammalian cell, and wherein dicer cleavageof said dsRNA preferentially results in an siRNA comprising said 3′ endof said antisense strand, thereby reducing expression of the target genein the mammal, wherein the antisense strand contains at least onethermally destabilizing nucleotide, where the at least one thermallydestabilizing nucleotide is in the seed region of the antisense strand(i.e. at position 2-9 of the 5′-end of the antisense strand), andwherein the dsRNA optionally further has at least one (e.g., one, two,three, four, five, six or all seven) of the following characteristics:(i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications;(ii) the antisense comprises 1, 2, 3, 4 or 5 phosphorothioateinternucleotide linkages; (iii) the sense strand is conjugated with aligand; (iv) the sense strand comprises 2, 3, 4 or 5 2′-fluoromodifications; (v) the sense strand comprises 1, 2, 3, 4 or 5phosphorothioate internucleotide linkages; and (vi) the dsRNA comprisesat least four 2′-fluoro modifications; and (vii) the dsRNA has a duplexregion of 12-29 nucleotide pairs in length.

In some embodiments, every nucleotide in the sense strand and antisensestrand of the dsRNA molecule may be modified. Each nucleotide may bemodified with the same or different modification which can include oneor more alteration of one or both of the non-linking phosphate oxygensand/or of one or more of the linking phosphate oxygens; alteration of aconstituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribosesugar; wholesale replacement of the phosphate moiety with “dephospho”linkers; modification or replacement of a naturally occurring base; andreplacement or modification of the ribose-phosphate backbone.

As nucleic acids are polymers of subunits, many of the modificationsoccur at a position which is repeated within a nucleic acid, e.g., amodification of a base, or a phosphate moiety, or a non-linking 0 of aphosphate moiety. In some cases the modification will occur at all ofthe subject positions in the nucleic acid but in many cases it will not.By way of example, a modification may only occur at a 3′ or 5′ terminalposition, may only occur in a terminal region, e.g., at a position on aterminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of astrand. A modification may occur in a double strand region, a singlestrand region, or in both. A modification may occur only in the doublestrand region of a RNA or may only occur in a single strand region of aRNA. E.g., a phosphorothioate modification at a non-linking 0 positionmay only occur at one or both termini, may only occur in a terminalregion, e.g., at a position on a terminal nucleotide or in the last 2,3, 4, 5, or 10 nucleotides of a strand, or may occur in double strandand single strand regions, particularly at termini. The 5′ end or endscan be phosphorylated.

It may be possible, e.g., to enhance stability, to include particularbases in overhangs, or to include modified nucleotides or nucleotidesurrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, orin both. E.g., it can be desirable to include purine nucleotides inoverhangs. In some embodiments all or some of the bases in a 3′ or 5′overhang may be modified, e.g., with a modification described herein.Modifications can include, e.g., the use of modifications at the 2′position of the ribose sugar with modifications that are known in theart, e.g., the use of deoxyribonucleotides, 2′-deoxy-2′-fluoro (2′-F) or2′-O-methyl modified instead of the ribosugar of the nucleobase, andmodifications in the phosphate group, e.g., phosphorothioatemodifications. Overhangs need not be homologous with the targetsequence.

In some embodiments, each residue of the sense strand and antisensestrand is independently modified with LNA, HNA, CeNA, 2′-methoxyethyl,2′-O-methyl, 2′-O-allyl, 2′-C-allyl, 2′-deoxy, or 2′-fluoro. The strandscan contain more than one modification. In some embodiments, eachresidue of the sense strand and antisense strand is independentlymodified with 2′-O-methyl or 2′-fluoro. It is to be understood thatthese modifications are in addition to the at least one thermallydestabilizing modification of the duplex present in the anti sensestrand.

At least two different modifications are typically present on the sensestrand and antisense strand. Those two modifications may be the2′-deoxy, 2′-O-methyl or 2′-fluoro modifications, acyclic nucleotides orothers. In some embodiments, the sense strand and antisense strand eachcomprises two differently modified nucleotides selected from 2′-O-methylor 2′-deoxy. In some embodiments, each residue of the sense strand andantisense strand is independently modified with 2′-O-methyl nucleotide,2′-deoxy nucleotide, 2″-deoxy-2′-fluoro nucleotide,2′-O—N-methylacetamido (2′-O-NMA) nucleotide, a2′-O-dimethylaminoethoxyethyl (2′-O-DMAEOE) nucleotide, 2′-O-aminopropyl(2′-O-AP) nucleotide, or 2′-ara-F nucleotide. Again, it is to beunderstood that these modifications are in addition to the at least onethermally destabilizing modification of the duplex present in theantisense strand.

In some embodiments, the dsRNA molecule of the disclosure comprisesmodifications of an alternating pattern, particular in the B1, B2, B3,B1′, B2′, B3′, B4′ regions. The term “alternating motif” or “alternativepattern” as used herein refers to a motif having one or moremodifications, each modification occurring on alternating nucleotides ofone strand. The alternating nucleotide may refer to one per every othernucleotide or one per every three nucleotides, or a similar pattern. Forexample, if A, B and C each represent one type of modification to thenucleotide, the alternating motif can be “ABABABABABAB . . . ,”“AABBAABBAABB . . . ,” “AABAABAABAAB . . . ,” “AAABAAABAAAB . . . ,”“AAABBBAAABBB . . . ,” or “ABCABCABCABC . . . ,” etc. The type ofmodifications contained in the alternating motif may be the same ordifferent. For example, if A, B, C, D each represent one type ofmodification on the nucleotide, the alternating pattern, i.e.,modifications on every other nucleotide, may be the same, but each ofthe sense strand or antisense strand can be selected from severalpossibilities of modifications within the alternating motif such as“ABABAB . . . ”, “ACACAC . . . ” “BDBDBD . . . ” or “CDCDCD . . . ,”etc.

In some embodiments, the dsRNA molecule of the disclosure comprises themodification pattern for the alternating motif on the sense strandrelative to the modification pattern for the alternating motif on theantisense strand is shifted. The shift may be such that the modifiedgroup of nucleotides of the sense strand corresponds to a differentlymodified group of nucleotides of the antisense strand and vice versa.For example, the sense strand when paired with the antisense strand inthe dsRNA duplex, the alternating motif in the sense strand may startwith “ABABAB” from 5′-3′ of the strand and the alternating motif in theantisense strand may start with “BABABA” from 3′-5′ of the strand withinthe duplex region. As another example, the alternating motif in thesense strand may start with “AABBAABB” from 5′-3′ of the strand and thealternating motif in the antisense strand may start with “BBAABBAA” from3′-5′ of the strand within the duplex region, so that there is acomplete or partial shift of the modification patterns between the sensestrand and the antisense strand.

The dsRNA molecule of the disclosure may further comprise at least onephosphorothioate or methylphosphonate internucleotide linkage. Thephosphorothioate or methylphosphonate internucleotide linkagemodification may occur on any nucleotide of the sense strand orantisense strand or both in any position of the strand. For instance,the internucleotide linkage modification may occur on every nucleotideon the sense strand and/or antisense strand; each internucleotidelinkage modification may occur in an alternating pattern on the sensestrand or antisense strand; or the sense strand or antisense strandcomprises both internucleotide linkage modifications in an alternatingpattern. The alternating pattern of the internucleotide linkagemodification on the sense strand may be the same or different from theantisense strand, and the alternating pattern of the internucleotidelinkage modification on the sense strand may have a shift relative tothe alternating pattern of the internucleotide linkage modification onthe antisense strand.

In some embodiments, the dsRNA molecule comprises the phosphorothioateor methylphosphonate internucleotide linkage modification in theoverhang region. For example, the overhang region comprises twonucleotides having a phosphorothioate or methylphosphonateinternucleotide linkage between the two nucleotides. Internucleotidelinkage modifications also may be made to link the overhang nucleotideswith the terminal paired nucleotides within duplex region. For example,at least 2, 3, 4, or all the overhang nucleotides may be linked throughphosphorothioate or methylphosphonate internucleotide linkage, andoptionally, there may be additional phosphorothioate ormethylphosphonate internucleotide linkages linking the overhangnucleotide with a paired nucleotide that is next to the overhangnucleotide. For instance, there may be at least two phosphorothioateinternucleotide linkages between the terminal three nucleotides, inwhich two of the three nucleotides are overhang nucleotides, and thethird is a paired nucleotide next to the overhang nucleotide.Preferably, these terminal three nucleotides may be at the 3′-end of theantisense strand.

In some embodiments, the sense strand of the dsRNA molecule comprises1-10 blocks of two to ten phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one ofthe phosphorothioate or methylphosphonate internucleotide linkages isplaced at any position in the oligonucleotide sequence and the saidsense strand is paired with an antisense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of two phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, or 18 phosphate internucleotide linkages,wherein one of the phosphorothioate or methylphosphonate internucleotidelinkages is placed at any position in the oligonucleotide sequence andthe said antisense strand is paired with a sense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of three phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15 or 16 phosphate internucleotide linkages, wherein one ofthe phosphorothioate or methylphosphonate internucleotide linkages isplaced at any position in the oligonucleotide sequence and the saidantisense strand is paired with a sense strand comprising anycombination of phosphorothioate, methylphosphonate and phosphateinternucleotide linkages or an antisense strand comprising eitherphosphorothioate or methylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of four phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11,12, 13 or 14 phosphate internucleotide linkages, wherein one of thephosphorothioate or methylphosphonate internucleotide linkages is placedat any position in the oligonucleotide sequence and the said antisensestrand is paired with a sense strand comprising any combination ofphosphorothioate, methylphosphonate and phosphate internucleotidelinkages or an antisense strand comprising either phosphorothioate ormethylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of five phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11or 12 phosphate internucleotide linkages, wherein one of thephosphorothioate or methylphosphonate internucleotide linkages is placedat any position in the oligonucleotide sequence and the said antisensestrand is paired with a sense strand comprising any combination ofphosphorothioate, methylphosphonate and phosphate internucleotidelinkages or an antisense strand comprising either phosphorothioate ormethylphosphonate or phosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of six phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10phosphate internucleotide linkages, wherein one of the phosphorothioateor methylphosphonate internucleotide linkages is placed at any positionin the oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of seven phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5, 6, 7 or 8 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of eight phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3, 4, 5 or 6 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In some embodiments, the antisense strand of the dsRNA moleculecomprises two blocks of nine phosphorothioate or methylphosphonateinternucleotide linkages separated by 1, 2, 3 or 4 phosphateinternucleotide linkages, wherein one of the phosphorothioate ormethylphosphonate internucleotide linkages is placed at any position inthe oligonucleotide sequence and the said antisense strand is pairedwith a sense strand comprising any combination of phosphorothioate,methylphosphonate and phosphate internucleotide linkages or an antisensestrand comprising either phosphorothioate or methylphosphonate orphosphate linkage.

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one or more phosphorothioate or methylphosphonateinternucleotide linkage modification within 1-10 of the terminiposition(s) of the sense and/or antisense strand. For example, at least2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked throughphosphorothioate or methylphosphonate internucleotide linkage at one endor both ends of the sense and/or antisense strand.

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one or more phosphorothioate or methylphosphonateinternucleotide linkage modification within 1-10 of the internal regionof the duplex of each of the sense and/or antisense strand. For example,at least 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotides may be linked throughphosphorothioate methylphosphonate internucleotide linkage at position8-16 of the duplex region counting from the 5′-end of the sense strand;the dsRNA molecule can optionally further comprise one or morephosphorothioate or methylphosphonate internucleotide linkagemodification within 1-10 of the termini position(s).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one to five phosphorothioate or methylphosphonateinternucleotide linkage modification(s) within position 1-5 and one tofive phosphorothioate or methylphosphonate internucleotide linkagemodification(s) within position 18-23 of the sense strand (counting fromthe 5′-end), and one to five phosphorothioate or methylphosphonateinternucleotide linkage modification at positions 1 and 2 and one tofive within positions 18-23 of the antisense strand (counting from the5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modificationwithin position 1-5 and one phosphorothioate or methylphosphonateinternucleotide linkage modification within position 18-23 of the sensestrand (counting from the 5′-end), and one phosphorothioateinternucleotide linkage modification at positions 1 and 2 and twophosphorothioate or methylphosphonate internucleotide linkagemodifications within positions 18-23 of the antisense strand (countingfrom the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 and two phosphorothioate internucleotide linkagemodifications within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 and two phosphorothioate internucleotide linkagemodifications within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modificationwithin position 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modificationwithin position 1-5 and one within position 18-23 of the sense strand(counting from the 5′-end), and two phosphorothioate internucleotidelinkage modification at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modificationwithin position 1-5 (counting from the 5′-end) of the sense strand, andtwo phosphorothioate internucleotide linkage modifications at positions1 and 2 and one phosphorothioate internucleotide linkage modificationwithin positions 18-23 of the antisense strand (counting from the5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 (counting from the 5′-end) of the sense strand, andone phosphorothioate internucleotide linkage modification at positions 1and 2 and two phosphorothioate internucleotide linkage modificationswithin positions 18-23 of the antisense strand (counting from the5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 and one within position 18-23 of the sense strand(counting from the 5′-end), and two phosphorothioate internucleotidelinkage modifications at positions 1 and 2 and one phosphorothioateinternucleotide linkage modification within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and two phosphorothioate internucleotide linkagemodifications at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modificationswithin position 1-5 and one phosphorothioate internucleotide linkagemodification within position 18-23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and 2 and two phosphorothioateinternucleotide linkage modifications within positions 18-23 of theantisense strand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modifications atposition 1 and 2, and two phosphorothioate internucleotide linkagemodifications at position 20 and 21 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and one at position 21 of the antisensestrand (counting from the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modification atposition 1, and one phosphorothioate internucleotide linkagemodification at position 21 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications at positions 20 and 21 the antisense strand (counting fromthe 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modifications atposition 1 and 2, and two phosphorothioate internucleotide linkagemodifications at position 21 and 22 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and one phosphorothioate internucleotidelinkage modification at position 21 of the antisense strand (countingfrom the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modification atposition 1, and one phosphorothioate internucleotide linkagemodification at position 21 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications at positions 21 and 22 the antisense strand (counting fromthe 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises two phosphorothioate internucleotide linkage modifications atposition 1 and 2, and two phosphorothioate internucleotide linkagemodifications at position 22 and 23 of the sense strand (counting fromthe 5′-end), and one phosphorothioate internucleotide linkagemodification at positions 1 and one phosphorothioate internucleotidelinkage modification at position 21 of the antisense strand (countingfrom the 5′-end).

In some embodiments, the dsRNA molecule of the disclosure furthercomprises one phosphorothioate internucleotide linkage modification atposition 1, and one phosphorothioate internucleotide linkagemodification at position 21 of the sense strand (counting from the5′-end), and two phosphorothioate internucleotide linkage modificationsat positions 1 and 2 and two phosphorothioate internucleotide linkagemodifications at positions 23 and 23 the antisense strand (counting fromthe 5′-end).

In some embodiments, compound of the disclosure comprises a pattern ofbackbone chiral centers. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 5 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 6 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 7 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 8 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 9 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 10 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 11 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 12 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 13 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 14 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 15 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 16 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 17 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 18 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises at least 19 internucleotidic linkagesin the Sp configuration. In some embodiments, a common pattern ofbackbone chiral centers comprises no more than 8 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 7 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 6 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 5 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 4 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 3 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 2 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 1 internucleotidiclinkages in the Rp configuration. In some embodiments, a common patternof backbone chiral centers comprises no more than 8 internucleotidiclinkages which are not chiral (as a non-limiting example, aphosphodiester). In some embodiments, a common pattern of backbonechiral centers comprises no more than 7 internucleotidic linkages whichare not chiral. In some embodiments, a common pattern of backbone chiralcenters comprises no more than 6 internucleotidic linkages which are notchiral. In some embodiments, a common pattern of backbone chiral centerscomprises no more than 5 internucleotidic linkages which are not chiral.In some embodiments, a common pattern of backbone chiral centerscomprises no more than 4 internucleotidic linkages which are not chiral.In some embodiments, a common pattern of backbone chiral centerscomprises no more than 3 internucleotidic linkages which are not chiral.In some embodiments, a common pattern of backbone chiral centerscomprises no more than 2 internucleotidic linkages which are not chiral.In some embodiments, a common pattern of backbone chiral centerscomprises no more than 1 internucleotidic linkages which are not chiral.In some embodiments, a common pattern of backbone chiral centerscomprises at least 10 internucleotidic linkages in the Sp configuration,and no more than 8 internucleotidic linkages which are not chiral. Insome embodiments, a common pattern of backbone chiral centers comprisesat least 11 internucleotidic linkages in the Sp configuration, and nomore than 7 internucleotidic linkages which are not chiral. In someembodiments, a common pattern of backbone chiral centers comprises atleast 12 internucleotidic linkages in the Sp configuration, and no morethan 6 internucleotidic linkages which are not chiral. In someembodiments, a common pattern of backbone chiral centers comprises atleast 13 internucleotidic linkages in the Sp configuration, and no morethan 6 internucleotidic linkages which are not chiral. In someembodiments, a common pattern of backbone chiral centers comprises atleast 14 internucleotidic linkages in the Sp configuration, and no morethan 5 internucleotidic linkages which are not chiral. In someembodiments, a common pattern of backbone chiral centers comprises atleast 15 internucleotidic linkages in the Sp configuration, and no morethan 4 internucleotidic linkages which are not chiral. In someembodiments, the internucleotidic linkages in the Sp configuration areoptionally contiguous or not contiguous. In some embodiments, theinternucleotidic linkages in the Rp configuration are optionallycontiguous or not contiguous. In some embodiments, the internucleotidiclinkages which are not chiral are optionally contiguous or notcontiguous.

In some embodiments, compound of the disclosure comprises a block is astereochemistry block. In some embodiments, a block is an Rp block inthat each internucleotidic linkage of the block is Rp. In someembodiments, a 5′-block is an Rp block. In some embodiments, a 3′-blockis an Rp block. In some embodiments, a block is an Sp block in that eachinternucleotidic linkage of the block is Sp. In some embodiments, a5′-block is an Sp block. In some embodiments, a 3′-block is an Sp block.In some embodiments, provided oligonucleotides comprise both Rp and Spblocks. In some embodiments, provided oligonucleotides comprise one ormore Rp but no Sp blocks. In some embodiments, provided oligonucleotidescomprise one or more Sp but no Rp blocks. In some embodiments, providedoligonucleotides comprise one or more PO blocks wherein eachinternucleotidic linkage in a natural phosphate linkage.

In some embodiments, compound of the disclosure comprises a 5′-block isan Sp block wherein each sugar moiety comprises a 2′-F modification. Insome embodiments, a 5′-block is an Sp block wherein each ofinternucleotidic linkage is a modified internucleotidic linkage and eachsugar moiety comprises a 2′-F modification. In some embodiments, a5′-block is an Sp block wherein each of internucleotidic linkage is aphosphorothioate linkage and each sugar moiety comprises a 2′-Fmodification. In some embodiments, a 5′-block comprises 4 or morenucleoside units. In some embodiments, a 5′-block comprises 5 or morenucleoside units. In some embodiments, a 5′-block comprises 6 or morenucleoside units. In some embodiments, a 5′-block comprises 7 or morenucleoside units. In some embodiments, a 3′-block is an Sp block whereineach sugar moiety comprises a 2′-F modification. In some embodiments, a3′-block is an Sp block wherein each of internucleotidic linkage is amodified internucleotidic linkage and each sugar moiety comprises a 2′-Fmodification. In some embodiments, a 3′-block is an Sp block whereineach of internucleotidic linkage is a phosphorothioate linkage and eachsugar moiety comprises a 2′-F modification. In some embodiments, a3′-block comprises 4 or more nucleoside units. In some embodiments, a3′-block comprises 5 or more nucleoside units. In some embodiments, a3′-block comprises 6 or more nucleoside units. In some embodiments, a3′-block comprises 7 or more nucleoside units.

In some embodiments, compound of the disclosure comprises a type ofnucleoside in a region or an oligonucleotide is followed by a specifictype of internucleotidic linkage, e.g., natural phosphate linkage,modified internucleotidic linkage, Rp chiral internucleotidic linkage,Sp chiral internucleotidic linkage, etc. In some embodiments, A isfollowed by Sp. In some embodiments, A is followed by Rp. In someembodiments, A is followed by natural phosphate linkage (PO). In someembodiments, U is followed by Sp. In some embodiments, U is followed byRp. In some embodiments, U is followed by natural phosphate linkage(PO). In some embodiments, C is followed by Sp. In some embodiments, Cis followed by Rp. In some embodiments, C is followed by naturalphosphate linkage (PO). In some embodiments, G is followed by Sp. Insome embodiments, G is followed by Rp. In some embodiments, G isfollowed by natural phosphate linkage (PO). In some embodiments, C and Uare followed by Sp. In some embodiments, C and U are followed by Rp. Insome embodiments, C and U are followed by natural phosphate linkage(PO). In some embodiments, A and G are followed by Sp. In someembodiments, A and G are followed by

Rp.

In some embodiments, the antisense strand comprises phosphorothioateinternucleotide linkages between nucleotide positions 21 and 22, andbetween nucleotide positions 22 and 23, wherein the antisense strandcontains at least one thermally destabilizing modification of the duplexlocated in the seed region of the antisense strand (i.e., at position2-9 of the 5′-end of the antisense strand), and wherein the dsRNAoptionally further has at least one (e.g., one, two, three, four, five,six, seven or all eight) of the following characteristics: (i) theantisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) theantisense comprises 3, 4 or 5 phosphorothioate internucleotide linkages;(iii) the sense strand is conjugated with a ligand; (iv) the sensestrand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) the sensestrand comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotidelinkages; (vi) the dsRNA comprises at least four 2′-fluoromodifications; (vii) the dsRNA comprises a duplex region of 12-40nucleotide pairs in length; and (viii) the dsRNA has a blunt end at5′-end of the antisense strand. In some embodiments, the antisensestrand comprises phosphorothioate internucleotide linkages betweennucleotide positions 1 and 2, between nucleotide positions 2 and 3,between nucleotide positions 21 and 22, and between nucleotide positions22 and 23, wherein the antisense strand contains at least one thermallydestabilizing modification of the duplex located in the seed region ofthe antisense strand (i.e., at position 2-9 of the 5′-end of theantisense strand), and wherein the dsRNA optionally further has at leastone (e.g., one, two, three, four, five, six, seven or all eight) of thefollowing characteristics: (i) the antisense comprises 2, 3, 4, 5 or 62′-fluoro modifications; (ii) the sense strand is conjugated with aligand; (iii) the sense strand comprises 2, 3, 4 or 5 2′-fluoromodifications; (iv) the sense strand comprises 1, 2, 3, 4 or 5phosphorothioate internucleotide linkages; (v) the dsRNA comprises atleast four 2′-fluoro modifications; (vi) the dsRNA comprises a duplexregion of 12-40 nucleotide pairs in length; (vii) the dsRNA comprises aduplex region of 12-40 nucleotide pairs in length; and (viii) the dsRNAhas a blunt end at 5′-end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioateinternucleotide linkages between nucleotide positions 1 and 2, andbetween nucleotide positions 2 and 3, wherein the antisense strandcontains at least one thermally destabilizing modification of the duplexlocated in the seed region of the antisense strand (i.e., at position2-9 of the 5′-end of the antisense strand), and wherein the dsRNAoptionally further has at least one (e.g., one, two, three, four, five,six, seven or all eight) of the following characteristics: (i) theantisense comprises 2, 3, 4, 5 or 6 2′-fluoro modifications; (ii) theantisense comprises 1, 2, 3, 4 or 5 phosphorothioate internucleotidelinkages; (iii) the sense strand is conjugated with a ligand; (iv) thesense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (v) thesense strand comprises 3, 4 or 5 phosphorothioate internucleotidelinkages; (vi) the dsRNA comprises at least four 2′-fluoromodifications; (vii) the dsRNA comprises a duplex region of 12-40nucleotide pairs in length; and (viii) the dsRNA has a blunt end at5′-end of the antisense strand.

In some embodiments, the sense strand comprises phosphorothioateinternucleotide linkages between nucleotide positions 1 and 2, andbetween nucleotide positions 2 and 3, the antisense strand comprisesphosphorothioate internucleotide linkages between nucleotide positions 1and 2, between nucleotide positions 2 and 3, between nucleotidepositions 21 and 22, and between nucleotide positions 22 and 23, whereinthe antisense strand contains at least one thermally destabilizingmodification of the duplex located in the seed region of the antisensestrand (i.e., at position 2-9 of the 5′-end of the antisense strand),and wherein the dsRNA optionally further has at least one (e.g., one,two, three, four, five, six or all seven) of the followingcharacteristics: (i) the antisense comprises 2, 3, 4, 5 or 6 2′-fluoromodifications; (ii) the sense strand is conjugated with a ligand; (iii)the sense strand comprises 2, 3, 4 or 5 2′-fluoro modifications; (iv)the sense strand comprises 3, 4 or 5 phosphorothioate internucleotidelinkages; (v) the dsRNA comprises at least four 2′-fluoro modifications;(vi) the dsRNA comprises a duplex region of 12-40 nucleotide pairs inlength; and (vii) the dsRNA has a blunt end at 5′-end of the antisensestrand.

In some embodiments, the dsRNA molecule of the disclosure comprisesmismatch(es) with the target, within the duplex, or combinationsthereof. The mismatch can occur in the overhang region or the duplexregion. The base pair can be ranked on the basis of their propensity topromote dissociation or melting (e.g., on the free energy of associationor dissociation of a particular pairing, the simplest approach is toexamine the pairs on an individual pair basis, though next neighbor orsimilar analysis can also be used). In terms of promoting dissociation:A:U is preferred over G:C; G:U is preferred over G:C; and I:C ispreferred over G:C (I=inosine). Mismatches, e.g., non-canonical or otherthan canonical pairings (as described elsewhere herein) are preferredover canonical (A:T, A:U, G:C) pairings; and pairings which include auniversal base are preferred over canonical pairings.

In some embodiments, the dsRNA molecule of the disclosure comprises atleast one of the first 1, 2, 3, 4, or 5 base pairs within the duplexregions from the 5′-end of the antisense strand can be chosenindependently from the group of: A:U, G:U, I:C, and mismatched pairs,e.g., non-canonical or other than canonical pairings or pairings whichinclude a universal base, to promote the dissociation of the antisensestrand at the 5′-end of the duplex.

In some embodiments, the nucleotide at the 1 position within the duplexregion from the 5′-end in the antisense strand is selected from thegroup consisting of A, dA, dU, U, and dT. Alternatively, at least one ofthe first 1, 2 or 3 base pair within the duplex region from the 5′-endof the antisense strand is an AU base pair. For example, the first basepair within the duplex region from the 5′-end of the antisense strand isan AU base pair.

It was found that introducing 4′-modified and/or 5′-modified nucleotideto the 3′-end of a phosphodiester (PO), phosphorothioate (PS), and/orphosphorodithioate (PS2) linkage of a dinucleotide at any position ofsingle stranded or double stranded oligonucleotide can exert stericeffect to the internucleotide linkage and, hence, protecting orstabilizing it against nucleases.

In some embodiments, 5′-modified nucleoside is introduced at the 3′-endof a dinucleotide at any position of single stranded or double strandedsiRNA. For instance, a 5′-alkylated nucleoside may be introduced at the3′-end of a dinucleotide at any position of single stranded or doublestranded siRNA. The alkyl group at the 5′ position of the ribose sugarcan be racemic or chirally pure R or S isomer. An exemplary 5′-alkylatednucleoside is 5′-methyl nucleoside. The 5′-methyl can be either racemicor chirally pure R or S isomer.

In some embodiments, 4′-modified nucleoside is introduced at the 3′-endof a dinucleotide at any position of single stranded or double strandedsiRNA. For instance, a 4′-alkylated nucleoside may be introduced at the3′-end of a dinucleotide at any position of single stranded or doublestranded siRNA. The alkyl group at the 4′ position of the ribose sugarcan be racemic or chirally pure R or S isomer. An exemplary 4′-alkylatednucleoside is 4′-methyl nucleoside. The 4′-methyl can be either racemicor chirally pure R or S isomer. Alternatively, a 4′-O-alkylatednucleoside may be introduced at the 3′-end of a dinucleotide at anyposition of single stranded or double stranded siRNA. The 4′-O-alkyl ofthe ribose sugar can be racemic or chirally pure R or S isomer. Anexemplary 4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The4′-O-methyl can be either racemic or chirally pure R or S isomer.

In some embodiments, 5′-alkylated nucleoside is introduced at anyposition on the sense strand or antisense strand of a dsRNA, and suchmodification maintains or improves potency of the dsRNA. The 5′-alkylcan be either racemic or chirally pure R or S isomer. An exemplary5′-alkylated nucleoside is 5′-methyl nucleoside. The 5′-methyl can beeither racemic or chirally pure R or S isomer.

In some embodiments, 4′-alkylated nucleoside is introduced at anyposition on the sense strand or antisense strand of a dsRNA, and suchmodification maintains or improves potency of the dsRNA. The 4′-alkylcan be either racemic or chirally pure R or S isomer. An exemplary4′-alkylated nucleoside is 4′-methyl nucleoside. The 4′-methyl can beeither racemic or chirally pure R or S isomer.

In some embodiments, 4′-O-alkylated nucleoside is introduced at anyposition on the sense strand or antisense strand of a dsRNA, and suchmodification maintains or improves potency of the dsRNA. The 5′-alkylcan be either racemic or chirally pure R or S isomer. An exemplary4′-O-alkylated nucleoside is 4′-O-methyl nucleoside. The 4′-O-methyl canbe either racemic or chirally pure R or S isomer.

In some embodiments, the dsRNA molecule of the disclosure can comprise2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P=0 or P═S). Forexample, the 2′-5′ linkages modifications can be used to promotenuclease resistance or to inhibit binding of the sense to the antisensestrand, or can be used at the 5′ end of the sense strand to avoid sensestrand activation by RISC.

In another embodiment, the dsRNA molecule of the disclosure can compriseL sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe). Forexample, these L sugars modifications can be used to promote nucleaseresistance or to inhibit binding of the sense to the antisense strand,or can be used at the 5′ end of the sense strand to avoid sense strandactivation by RISC.

Various publications describe multimeric siRNA which can all be usedwith the dsRNA of the disclosure. Such publications includeWO2007/091269, U.S. Pat. No. 7,858,769, WO2010/141511, WO2007/117686,WO2009/014887 and WO2011/031520 which are hereby incorporated by theirentirely.

The dsRNA molecule that contains conjugations of one or morecarbohydrate moieties to a dsRNA molecule can optimize one or moreproperties of the dsRNA molecule. In many cases, the carbohydrate moietywill be attached to a modified subunit of the dsRNA molecule. E.g., theribose sugar of one or more ribonucleotide subunits of a dsRNA moleculecan be replaced with another moiety, e.g., a non-carbohydrate(preferably cyclic) carrier to which is attached a carbohydrate ligand.A ribonucleotide subunit in which the ribose sugar of the subunit hasbeen so replaced is referred to herein as a ribose replacementmodification subunit (RRMS). A cyclic carrier may be a carbocyclic ringsystem, i.e., all ring atoms are carbon atoms, or a heterocyclic ringsystem, i.e., one or more ring atoms may be a heteroatom, e.g.,nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ringsystem, or may contain two or more rings, e.g. fused rings. The cycliccarrier may be a fully saturated ring system, or it may contain one ormore double bonds.

The ligand may be attached to the polynucleotide via a carrier. Thecarriers include (i) at least one “backbone attachment point,”preferably two “backbone attachment points” and (ii) at least one“tethering attachment point.” A “backbone attachment point” as usedherein refers to a functional group, e.g. a hydroxyl group, orgenerally, a bond available for, and that is suitable for incorporationof the carrier into the backbone, e.g., the phosphate, or modifiedphosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A“tethering attachment point” (TAP) in some embodiments refers to aconstituent ring atom of the cyclic carrier, e.g., a carbon atom or aheteroatom (distinct from an atom which provides a backbone attachmentpoint), that connects a selected moiety. The moiety can be, e.g., acarbohydrate, e.g. monosaccharide, disaccharide, trisaccharide,tetrasaccharide, oligosaccharide and polysaccharide. Optionally, theselected moiety is connected by an intervening tether to the cycliccarrier. Thus, the cyclic carrier will often include a functional group,e.g., an amino group, or generally, provide a bond, that is suitable forincorporation or tethering of another chemical entity, e.g., a ligand tothe constituent ring.

In one embodiment the dsRNA molecule of the disclosure is conjugated toa ligand via a carrier, wherein the carrier can be cyclic group oracyclic group; preferably, the cyclic group is selected frompyrrolidinyl, pyrazolinyl, pyrazolidinyl, imidazolinyl, imidazolidinyl,piperidinyl, piperazinyl, [1,3]dioxolane, oxazolidinyl, isoxazolidinyl,morpholinyl, thiazolidinyl, isothiazolidinyl, quinoxalinyl,pyridazinonyl, tetrahydrofuryl and decalin; preferably, the acyclicgroup is selected from serinol backbone or diethanolamine backbone.

The double-stranded RNA (dsRNA) agent of the disclosure may optionallybe conjugated to one or more ligands. The ligand can be attached to thesense strand, antisense strand or both strands, at the 3′-end, 5′-end orboth ends. For instance, the ligand may be conjugated to the sensestrand, in particular, the 3′-end of the sense strand.

In some embodiments dsRNA molecules of the disclosure are 5′phosphorylated or include a phosphoryl analog at the 5′ prime terminus.5′-phosphate modifications include those which are compatible with RISCmediated gene silencing. Suitable modifications include:5′-monophosphate ((HO)₂(O)P—O-5′); 5′-diphosphate((HO)₂(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylatedor non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-adenosine cap (Appp), and any modified or unmodified nucleotide capstructure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′);5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O-5′);5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′),5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination ofoxygen/sulfur replaced monophosphate, diphosphate and triphosphates(e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.),5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O-5′),5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc.,e.g. RP(OH)(O)—O-5′-, 5′-alkenylphosphonates (i.e. vinyl, substitutedvinyl), (OH)₂(O)P-5′-CH2-), 5′-alkyletherphosphonates(R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g.RP(OH)(O)—O-5′-). In one example, the modification can in placed in theantisense strand of a dsRNA molecule.

F. Linkers

In some embodiments, the conjugate or ligand described herein can beattached to a RNAi agent oligonucleotide with various linkers that canbe cleavable or non cleavable.

The term “linker” or “linking group” means an organic moiety thatconnects two parts of a compound, e.g., covalently attaches two parts ofa compound. Linkers typically comprise a direct bond or an atom such asoxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO₂, SO₂NH or achain of atoms, such as, but not limited to, substituted orunsubstituted alkyl, substituted or unsubstituted alkenyl, substitutedor unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl,heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl,heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl,heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl,alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl,alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl,alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl,alkylheteroarylalkenyl, alkylheteroarylalkynyl, alkenylheteroarylalkyl,alkenylheteroarylalkenyl, alkenylheteroarylalkynyl,alkynylheteroarylalkyl, alkynylheteroarylalkenyl,alkynylheteroarylalkynyl, alkylheterocyclylalkyl,alkylheterocyclylalkenyl, alkylhererocyclylalkynyl,alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl,alkenylheterocyclylalkynyl, alkynylheterocyclylalkyl,alkynylheterocyclylalkenyl, alkynylheterocyclylalkynyl, alkylaryl,alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl,alkynylhereroaryl, which one or more methylenes can be interrupted orterminated by O, S, S(O), SO₂, N(R8), C(O), substituted or unsubstitutedaryl, substituted or unsubstituted heteroaryl, substituted orunsubstituted heterocyclic; where R8 is hydrogen, acyl, aliphatic orsubstituted aliphatic. In one embodiment, the linker is between about1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17,8-17, 6-16, 7-17, or 8-16 atoms.

A cleavable linking group is one which is sufficiently stable outsidethe cell, but which upon entry into a target cell is cleaved to releasethe two parts the linker is holding together. In a preferred embodiment,the cleavable linking group is cleaved at least about 10 times, 20,times, 30 times, 40 times, 50 times, 60 times, 70 times, 80 times, 90times or more, or at least about 100 times faster in a target cell orunder a first reference condition (which can, e.g., be selected to mimicor represent intracellular conditions) than in the blood of a subject,or under a second reference condition (which can, e.g., be selected tomimic or represent conditions found in the blood or serum).

Cleavable linking groups are susceptible to cleavage agents, e.g., pH,redox potential or the presence of degradative molecules. Generally,cleavage agents are more prevalent or found at higher levels oractivities inside cells than in serum or blood. Examples of suchdegradative agents include: redox agents which are selected forparticular substrates or which have no substrate specificity, including,e.g., oxidative or reductive enzymes or reductive agents such asmercaptans, present in cells, that can degrade a redox cleavable linkinggroup by reduction; esterases; endosomes or agents that can create anacidic environment, e.g., those that result in a pH of five or lower;enzymes that can hydrolyze or degrade an acid cleavable linking group byacting as a general acid, peptidases (which can be substrate specific),and phosphatases.

A cleavable linkage group, such as a disulfide bond can be susceptibleto pH. The pH of human serum is 7.4, while the average intracellular pHis slightly lower, ranging from about 7.1-7.3. Endosomes have a moreacidic pH, in the range of 5.5-6.0, and lysosomes have an even moreacidic pH at around 5.0. Some linkers will have a cleavable linkinggroup that is cleaved at a preferred pH, thereby releasing a cationiclipid from the ligand inside the cell, or into the desired compartmentof the cell.

A linker can include a cleavable linking group that is cleavable by aparticular enzyme. The type of cleavable linking group incorporated intoa linker can depend on the cell to be targeted.

In general, the suitability of a candidate cleavable linking group canbe evaluated by testing the ability of a degradative agent (orcondition) to cleave the candidate linking group. It will also bedesirable to also test the candidate cleavable linking group for theability to resist cleavage in the blood or when in contact with othernon-target tissue. Thus, one can determine the relative susceptibilityto cleavage between a first and a second condition, where the first isselected to be indicative of cleavage in a target cell and the second isselected to be indicative of cleavage in other tissues or biologicalfluids, e.g., blood or serum. The evaluations can be carried out in cellfree systems, in cells, in cell culture, in organ or tissue culture, orin whole animals. It can be useful to make initial evaluations incell-free or culture conditions and to confirm by further evaluations inwhole animals. In preferred embodiments, useful candidate compounds arecleaved at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, orabout 100 times faster in the cell (or under in vitro conditionsselected to mimic intracellular conditions) as compared to blood orserum (or under in vitro conditions selected to mimic extracellularconditions).

i. Redox Cleavable Linking Groups

In one embodiment, a cleavable linking group is a redox cleavablelinking group that is cleaved upon reduction or oxidation. An example ofreductively cleavable linking group is a disulphide linking group(—S—S—). To determine if a candidate cleavable linking group is asuitable “reductively cleavable linking group,” or for example issuitable for use with a particular iRNA moiety and particular targetingagent one can look to methods described herein. For example, a candidatecan be evaluated by incubation with dithiothreitol (DTT), or otherreducing agent using reagents know in the art, which mimic the rate ofcleavage which would be observed in a cell, e.g., a target cell. Thecandidates can also be evaluated under conditions which are selected tomimic blood or serum conditions. In one, candidate compounds are cleavedby at most about 10% in the blood. In other embodiments, usefulcandidate compounds are degraded at least about 2, 4, 10, 20, 30, 40,50, 60, 70, 80, 90, or about 100 times faster in the cell (or under invitro conditions selected to mimic intracellular conditions) as comparedto blood (or under in vitro conditions selected to mimic extracellularconditions). The rate of cleavage of candidate compounds can bedetermined using standard enzyme kinetics assays under conditions chosento mimic intracellular media and compared to conditions chosen to mimicextracellular media.

ii. Phosphate-based cleavable linking groups In another embodiment, acleavable linker comprises a phosphate-based cleavable linking group. Aphosphate-based cleavable linking group is cleaved by agents thatdegrade or hydrolyze the phosphate group. An example of an agent thatcleaves phosphate groups in cells are enzymes such as phosphatases incells. Examples of phosphate-based linking groups are —O—P(O)(ORk)-O—,—O—P(S)(ORk)-O—, —O—P(S)(SRk)-O—, —S—P(O)(ORk)-O—, —O—P(O)(ORk)-S—,—S—P(O)(ORk)-S—, —O—P(S)(ORk)-S—, —S—P(S)(ORk)-O—, —O—P(O)(Rk)-O—,—O—P(S)(Rk)-O—, —S—P(O)(Rk)-O—, —S—P(S)(Rk)-O—, —S—P(O)(Rk)-S—,—O—P(S)(Rk)-S—. Preferred embodiments are —O—P(O)(OH)—O—,—O—P(S)(OH)—O—, —O—P(S)(SH)—O—, —S—P(O)(OH)—O—, —O—P(O)(OH)—S—,—S—P(O)(OH)—S—, —O—P(S)(OH)—S—, —S—P(S)(OH)—O—, —O—P(O)(H)—O—,—O—P(S)(H)—O—, —S—P(O)(H)—O—, —S—P(S)(H)—O—, —S—P(O)(H)—S—,—O—P(S)(H)—S—. A preferred embodiment is —O—P(O)(OH)—O—. Thesecandidates can be evaluated using methods analogous to those describedabove.

iii. Acid Cleavable Linking Groups

In another embodiment, a cleavable linker comprises an acid cleavablelinking group. An acid cleavable linking group is a linking group thatis cleaved under acidic conditions. In preferred embodiments acidcleavable linking groups are cleaved in an acidic environment with a pHof about 6.5 or lower (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or lower),or by agents such as enzymes that can act as a general acid. In a cell,specific low pH organelles, such as endosomes and lysosomes can providea cleaving environment for acid cleavable linking groups. Examples ofacid cleavable linking groups include but are not limited to hydrazones,esters, and esters of amino acids. Acid cleavable groups can have thegeneral formula —C═NN—, C(O)O, or —OC(O). A preferred embodiment is whenthe carbon attached to the oxygen of the ester (the alkoxy group) is anaryl group, substituted alkyl group, or tertiary alkyl group such asdimethyl pentyl or t-butyl. These candidates can be evaluated usingmethods analogous to those described above.

iv. Ester-Based Linking Groups

In another embodiment, a cleavable linker comprises an ester-basedcleavable linking group. An ester-based cleavable linking group iscleaved by enzymes such as esterases and amidases in cells. Examples ofester-based cleavable linking groups include but are not limited toesters of alkylene, alkenylene and alkynylene groups. Ester cleavablelinking groups have the general formula —C(O)O—, or —OC(O)—. Thesecandidates can be evaluated using methods analogous to those describedabove.

v. Peptide-Based Cleaving Groups

In yet another embodiment, a cleavable linker comprises a peptide-basedcleavable linking group. A peptide-based cleavable linking group iscleaved by enzymes such as peptidases and proteases in cells.Peptide-based cleavable linking groups are peptide bonds formed betweenamino acids to yield oligopeptides (e.g., dipeptides, tripeptides etc.)and polypeptides. Peptide-based cleavable groups do not include theamide group (—C(O)NH—). The amide group can be formed between anyalkylene, alkenylene or alkynelene. A peptide bond is a special type ofamide bond formed between amino acids to yield peptides and proteins.The peptide based cleavage group is generally limited to the peptidebond (i.e., the amide bond) formed between amino acids yielding peptidesand proteins and does not include the entire amide functional group.Peptide-based cleavable linking groups have the general formula—NHCHRAC(O)NHCHRBC(O)—, where RA and RB are the R groups of the twoadjacent amino acids. These candidates can be evaluated using methodsanalogous to those described above.

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; 8,106,022, the entire contents of each of whichare hereby incorporated herein by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications can be incorporated in a single compound or even at asingle nucleoside within a RNAi agent. The present disclosure alsoincludes RNAi agents that are chimeric compounds.

“Chimeric” RNAi agents or “chimeras,” in the context of this disclosure,are RNAi agents, preferably dsRNAs, which contain two or more chemicallydistinct regions, each made up of at least one monomer unit, i.e., anucleotide in the case of a dsRNA compound. These RNAi agents typicallycontain at least one region wherein the RNA is modified so as to conferupon the RNAi agent increased resistance to nuclease degradation,increased cellular uptake, and/or increased binding affinity for thetarget nucleic acid. An additional region of the RNAi agent can serve asa substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids.By way of example, RNase H is a cellular endonuclease which cleaves theRNA strand of an RNA:DNA duplex. Activation of RNase H, therefore,results in cleavage of the RNA target, thereby greatly enhancing theefficiency of RNAi agent-mediated inhibition of gene expression.Consequently, comparable results can often be obtained with shorter RNAiagents when chimeric dsRNAs are used, compared to phosphorothioate deoxydsRNAs hybridizing to the same target region. Cleavage of the RNA targetcan be routinely detected by gel electrophoresis and, if necessary,associated nucleic acid hybridization techniques known in the art.

In certain instances, the RNA of a RNAi agent can be modified by anon-ligand group. A number of non-ligand molecules have been conjugatedto RNAi agents in order to enhance the activity, cellular distributionor cellular uptake of the RNAi agent, and procedures for performing suchconjugations are available in the scientific literature. Such non-ligandmoieties have included lipid moieties, such as cholesterol (Kubo, T. etal., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al.,Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan etal., Bioorg. Med. Chem. Lett., 1994, 4:1053), a thioether, e.g.,hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3:2765), athiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533), analiphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaraset al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990,259:327; Svinarchuk et al., Biochimie, 1993, 75:49), a phospholipid,e.g., di-hexadecyl-rac-glycerol or tri ethyl ammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction can be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

VI. Delivery of a RNAi Agent of the Disclosure

The delivery of a RNAi agent of the disclosure to a cell e.g., a cellwithin a subject, such as a human subject (e.g., a subject in needthereof, such as a subject having an APP-associated disorder, e.g., CAAand/or AD, e.g., EOFAD) can be achieved in a number of different ways.For example, delivery may be performed by contacting a cell with a RNAiagent of the disclosure either in vitro or in vivo. In vivo delivery mayalso be performed directly by administering a composition comprising aRNAi agent, e.g., a dsRNA, to a subject. Alternatively, in vivo deliverymay be performed indirectly by administering one or more vectors thatencode and direct the expression of the RNAi agent. These alternativesare discussed further below.

In general, any method of delivering a nucleic acid molecule (in vitroor in vivo) can be adapted for use with a RNAi agent of the disclosure(see e.g., Akhtar S. and Julian R L., (1992) Trends Cell. Biol.2(5):139-144 and WO94/02595, which are incorporated herein by referencein their entireties). For in vivo delivery, factors to consider in orderto deliver a RNAi agent include, for example, biological stability ofthe delivered agent, prevention of non-specific effects, andaccumulation of the delivered agent in the target tissue. Thenon-specific effects of a RNAi agent can be minimized by localadministration, for example, by direct injection or implantation into atissue or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that can otherwise beharmed by the agent or that can degrade the agent, and permits a lowertotal dose of the RNAi agent to be administered. Several studies haveshown successful knockdown of gene products when a RNAi agent isadministered locally. For example, intraocular delivery of a VEGF dsRNAby intravitreal injection in cynomolgus monkeys (Tolentino, M J. et al.,(2004) Retina 24:132-138) and subretinal injections in mice (Reich, S J.et al. (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J. et al. (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J. etal., (2006) Mol. Ther. 14:343-350; Li, S. et al., (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G. et al., (2004) Nucleic Acids32:e49; Tan, P H. et al. (2005) Gene Ther. 12:59-66; Makimura, H. et al.(2002) BMC Neurosci. 3:18; Shishkina, G T., et al. (2004) Neuroscience129:521-528; Thakker, E R., et al. (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al. (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A. et al.,(2006) Mol. Ther. 14:476-484; Zhang, X. et al., (2004) J. Biol. Chem.279:10677-10684; Bitko, V. et al., (2005) Nat. Med. 11:50-55). Foradministering a RNAi agent systemically for the treatment of a disease,the RNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the RNAi agent tothe target tissue and avoid undesirable off-target effects (e.g.,without wishing to be bound by theory, use of GNAs as described hereinhas been identified to destabilize the seed region of a dsRNA, resultingin enhanced preference of such dsRNAs for on-target effectiveness,relative to off-target effects, as such off-target effects aresignificantly weakened by such seed region destabilization). RNAi agentscan be modified by chemical conjugation to lipophilic groups such ascholesterol to enhance cellular uptake and prevent degradation. Forexample, a RNAi agent directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J. etal., (2004) Nature 432:173-178). Conjugation of a RNAi agent to anaptamer has been shown to inhibit tumor growth and mediate tumorregression in a mouse model of prostate cancer (McNamara, J O. et al.,(2006) Nat. Biotechnol. 24:1005-1015). In an alternative embodiment, theRNAi agent can be delivered using drug delivery systems such as ananoparticle, a dendrimer, a polymer, liposomes, or a cationic deliverysystem. Positively charged cationic delivery systems facilitate bindingof molecule RNAi agent (negatively charged) and also enhanceinteractions at the negatively charged cell membrane to permit efficientuptake of a RNAi agent by the cell. Cationic lipids, dendrimers, orpolymers can either be bound to a RNAi agent, or induced to form avesicle or micelle (see e.g., Kim S H. et al., (2008) Journal ofControlled Release 129(2):107-116) that encases a RNAi agent. Theformation of vesicles or micelles further prevents degradation of theRNAi agent when administered systemically. Methods for making andadministering cationic-RNAi agent complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al.(2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al. (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of RNAi agents include DOTAP (Sorensen, D R., et al (2003),supra; Verma, U N. et al., (2003), supra), Oligofectamine, “solidnucleic acid lipid particles” (Zimmermann, T S. et al., (2006) Nature441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther.12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091),polyethyleneimine (Bonnet M E. et al., (2008) Pharm. Res. August 16 Epubahead of print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659),Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), andpolyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans.35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In someembodiments, a RNAi agent forms a complex with cyclodextrin for systemicadministration. Methods for administration and pharmaceuticalcompositions of RNAi agents and cyclodextrins can be found in U.S. Pat.No. 7,427,605, which is herein incorporated by reference in itsentirety.

Certain aspects of the instant disclosure relate to a method of reducingthe expression of an APP target gene in a cell, comprising contactingsaid cell with the double-stranded RNAi agent of the disclosure. In oneembodiment, the cell is an extraheptic cell, optionally a CNS cell.

Another aspect of the disclosure relates to a method of reducing theexpression of an APP target gene in a subject, comprising administeringto the subject the double-stranded RNAi agent of the disclosure.

Another aspect of the disclosure relates to a method of treating asubject having a CNS disorder, comprising administering to the subject atherapeutically effective amount of the double-stranded APP-targetingRNAi agent of the disclosure, thereby treating the subject. ExemplaryCNS disorders that can be treated by the method of the disclosureinclude alzheimer, amyotrophic lateral schlerosis (ALS), frontotemporaldementia, huntington, Parkinson, spinocerebellar, prion, and lafora.

In one embodiment, the double-stranded RNAi agent is administeredintrathecally. By intrathecal administration of the double-stranded RNAiagent, the method can reduce the expression of an APP target gene in abrain or spine tissue, for instance, cortex, cerebellum, striatum,cervical spine, lumbar spine, and thoracic spine.

For ease of exposition the formulations, compositions and methods inthis section are discussed largely with regard to modified siRNAcompounds. It may be understood, however, that these formulations,compositions and methods can be practiced with other siRNA compounds,e.g., unmodified siRNA compounds, and such practice is within thedisclosure. A composition that includes a RNAi agent can be delivered toa subject by a variety of routes. Exemplary routes include: intrathecal,intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The RNAi agents of the disclosure can be incorporated intopharmaceutical compositions suitable for administration. Suchcompositions typically include one or more species of RNAi agent and apharmaceutically acceptable carrier. As used herein the language“pharmaceutically acceptable carrier” is intended to include any and allsolvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic and absorption delaying agents, and the like,compatible with pharmaceutical administration. The use of such media andagents for pharmaceutically active substances is well known in the art.Except insofar as any conventional media or agent is incompatible withthe active compound, use thereof in the compositions is contemplated.Supplementary active compounds can also be incorporated into thecompositions.

The pharmaceutical compositions of the present disclosure may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (including ophthalmic, vaginal, rectal,intranasal, transdermal), oral or parenteral. Parenteral administrationincludes intravenous drip, subcutaneous, intraperitoneal orintramuscular injection, or intrathecal or intraventricularadministration.

The route and site of administration may be chosen to enhance targeting.For example, to target muscle cells, intramuscular injection into themuscles of interest would be a logical choice. Lung cells might betargeted by administering the RNAi agent in aerosol form. The vascularendothelial cells could be targeted by coating a balloon catheter withthe RNAi agent and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules,suspensions or solutions in water, syrups, elixirs or non-aqueous media,tablets, capsules, lozenges, or troches. In the case of tablets,carriers that can be used include lactose, sodium citrate and salts ofphosphoric acid. Various disintegrants such as starch, and lubricatingagents such as magnesium stearate, sodium lauryl sulfate and talc, arecommonly used in tablets. For oral administration in capsule form,useful diluents are lactose and high molecular weight polyethyleneglycols. When aqueous suspensions are required for oral use, the nucleicacid compositions can be combined with emulsifying and suspendingagents. If desired, certain sweetening and/or flavoring agents can beadded.

Compositions for intrathecal or intraventricular administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueoussolutions which may also contain buffers, diluents and other suitableadditives. Intraventricular injection may be facilitated by anintraventricular catheter, for example, attached to a reservoir. Forintravenous use, the total concentration of solutes may be controlled torender the preparation isotonic.

In one embodiment, the administration of the siRNA compound, e.g., adouble-stranded siRNA compound, or ssiRNA compound, composition isparenteral, e.g., intravenous (e.g., as a bolus or as a diffusibleinfusion), intradermal, intraperitoneal, intramuscular, intrathecal,intraventricular, intracranial, subcutaneous, transmucosal, buccal,sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary,intranasal, urethral or ocular. Administration can be provided by thesubject or by another person, e.g., a health care provider. Themedication can be provided in measured doses or in a dispenser whichdelivers a metered dose. Selected modes of delivery are discussed inmore detail below.

Intrathecal Administration.

In one embodiment, the double-stranded RNAi agent is delivered byintrathecal injection (i.e. injection into the spinal fluid which bathesthe brain and spinal chord tissue). Intrathecal injection of RNAi agentsinto the spinal fluid can be performed as a bolus injection or viaminipumps which can be implanted beneath the skin, providing a regularand constant delivery of siRNA into the spinal fluid. The circulation ofthe spinal fluid from the choroid plexus, where it is produced, downaround the spinal chord and dorsal root ganglia and subsequently up pastthe cerebellum and over the cortex to the arachnoid granulations, wherethe fluid can exit the CNS, that, depending upon size, stability, andsolubility of the compounds injected, molecules delivered intrathecallycould hit targets throughout the entire CNS.

In some embodiments, the intrathecal administration is via a pump. Thepump may be a surgically implanted osmotic pump. In one embodiment, theosmotic pump is implanted into the subarachnoid space of the spinalcanal to facilitate intrathecal administration.

In some embodiments, the intrathecal administration is via anintrathecal delivery system for a pharmaceutical including a reservoircontaining a volume of the pharmaceutical agent, and a pump configuredto deliver a portion of the pharmaceutical agent contained in thereservoir. More details about this intrathecal delivery system may befound in PCT/US2015/013253, filed on Jan. 28, 2015, which isincorporated by reference in its entirety.

The amount of intrathecally injected RNAi agents may vary from onetarget gene to another target gene and the appropriate amount that hasto be applied may have to be determined individually for each targetgene. Typically, this amount ranges between 10 μg to 2 mg, preferably 50μg to 1500 more preferably 100 μg to 1000 μg.

A. Vector Encoded RNAi Agents of the Disclosure

RNAi agents targeting the APP gene can be expressed from transcriptionunits inserted into DNA or RNA vectors (see, e.g., Couture, A, et al.,TIG. (1996), 12:5-10; Skillern, A., et al., International PCTPublication No. WO 00/22113, Conrad, International PCT Publication No.WO 00/22114, and Conrad, U.S. Pat. No. 6,054,299). Expression can betransient (on the order of hours to weeks) or sustained (weeks to monthsor longer), depending upon the specific construct used and the targettissue or cell type. These transgenes can be introduced as a linearconstruct, a circular plasmid, or a viral vector, which can be anintegrating or non-integrating vector. The transgene can also beconstructed to permit it to be inherited as an extrachromosomal plasmid(Gassmann, et al., (1995) Proc. Natl. Acad. Sci. USA 92:1292).

The individual strand or strands of a RNAi agent can be transcribed froma promoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as invertedrepeat polynucleotides joined by a linker polynucleotide sequence suchthat the dsRNA has a stem and loop structure.

RNAi agent expression vectors are generally DNA plasmids or viralvectors. Expression vectors compatible with eukaryotic cells, preferablythose compatible with vertebrate cells, can be used to producerecombinant constructs for the expression of a RNAi agent as describedherein. Eukaryotic cell expression vectors are well known in the art andare available from a number of commercial sources. Typically, suchvectors are provided containing convenient restriction sites forinsertion of the desired nucleic acid segment. Delivery of RNAi agentexpressing vectors can be systemic, such as by intravenous orintramuscular administration, by administration to target cellsex-planted from the patient followed by reintroduction into the patient,or by any other means that allows for introduction into a desired targetcell.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct canbe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of a RNAiagent will generally require regulatory elements, e.g., promoters,enhancers, etc., to ensure the expression of the RNAi agent in targetcells. Other aspects to consider for vectors and constructs are known inthe art.

VII. Pharmaceutical Compositions of the Disclosure

The present disclosure also includes pharmaceutical compositions andformulations which include the RNAi agents of the disclosure. In oneembodiment, provided herein are pharmaceutical compositions containing aRNAi agent, as described herein, and a pharmaceutically acceptablecarrier. The pharmaceutical compositions containing the RNAi agent areuseful for treating a disease or disorder associated with the expressionor activity of an APP gene, e.g., an APP-associated disease, e.g., CAAor AD, e.g., EOFAD.

Such pharmaceutical compositions are formulated based on the mode ofdelivery. One example is compositions that are formulated for systemicadministration via parenteral delivery, e.g., by intravenous (IV),intramuscular (IM), or for subcutaneous (subQ) delivery. Another exampleis compositions that are formulated for direct delivery into the CNS,e.g., by intrathecal or intravitreal routes of injection, optionally byinfusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions of the disclosure may be administered indosages sufficient to inhibit expression of an APP gene. In general, asuitable dose of a RNAi agent of the disclosure will be in the range ofabout 0.001 to about 200.0 milligrams per kilogram body weight of therecipient per day, generally in the range of about 1 to 50 mg perkilogram body weight per day. Typically, a suitable dose of a RNAi agentof the disclosure will be in the range of about 0.1 mg/kg to about 5.0mg/kg, preferably about 0.3 mg/kg and about 3.0 mg/kg.

A repeat-dose regimen may include administration of a therapeutic amountof a RNAi agent on a regular basis, such as bi-monthly or monthly toonce a year. In certain embodiments, the RNAi agent is administeredabout once per month to about once per quarter (i.e., about once everythree months).

After an initial treatment regimen, the treatments can be administeredon a less frequent basis.

The dosage unit can be compounded for delivery over an extended period,e.g., using a conventional sustained release formulation which providessustained release of the RNAi agent over an extended period. Sustainedrelease formulations are well known in the art and are particularlyuseful for delivery of agents at a particular site, such as could beused with the agents of the present disclosure. In this embodiment, thedosage unit contains a corresponding multiple of, e.g., a monthlydose.

In other embodiments, a single dose of the pharmaceutical compositionscan be long lasting, such that subsequent doses are administered at notmore than 1, 2, 3, or 4 or more week intervals. In some embodiments ofthe disclosure, a single dose of the pharmaceutical compositions of thedisclosure is administered once per week. In other embodiments of thedisclosure, a single dose of the pharmaceutical compositions of thedisclosure is administered bi-monthly.

The skilled artisan will appreciate that certain factors can influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual RNAi agents encompassed by thedisclosure can be made using conventional methodologies or on the basisof in vivo testing using an appropriate animal model, as describedelsewhere herein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as APP-associated disordersthat would benefit from reduction in the expression of APP. Such modelscan be used for in vivo testing of RNAi agents, as well as fordetermining a therapeutically effective dose. Suitable mouse models areknown in the art and include, for example, the AD and/or CAA modelsdescribed elsewhere herein.

The pharmaceutical compositions of the present disclosure can beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration can be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The RNAi agents can be delivered in a manner to target a particulartissue, such as the CNS (e.g., neuronal, glial and/or vascular tissue ofthe brain).

Pharmaceutical compositions and formulations for topical administrationcan include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like can be necessary or desirable. Coated condoms, gloves and thelike can also be useful. Suitable topical formulations include those inwhich the RNAi agents featured in the disclosure are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). RNAi agents featured inthe disclosure can be encapsulated within liposomes or can formcomplexes thereto, in particular to cationic liposomes. Alternatively,RNAi agents can be complexed to lipids, in particular to cationiclipids. Suitable fatty acids and esters include but are not limited toarachidonic acid, oleic acid, eicosanoic acid, lauric acid, caprylicacid, capric acid, myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein, dilaurin,glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine,an acylcholine, or a C₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM),monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.Topical formulations are described in detail in U.S. Pat. No. 6,747,014,which is incorporated herein by reference.

A. RNAi Agent Formulations Comprising Membranous Molecular Assemblies

A RNAi agent for use in the compositions and methods of the disclosurecan be formulated for delivery in a membranous molecular assembly, e.g.,a liposome or a micelle. As used herein, the term “liposome” refers to avesicle composed of amphiphilic lipids arranged in at least one bilayer,e.g., one bilayer or a plurality of bilayers. Liposomes includeunilamellar and multilamellar vesicles that have a membrane formed froma lipophilic material and an aqueous interior. The aqueous portioncontains the RNAi agent composition. The lipophilic material isolatesthe aqueous interior from an aqueous exterior, which typically does notinclude the RNAi agent composition, although in some examples, it may.Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomal bilayer fuses with bilayer of the cellular membranes. Asthe merging of the liposome and cell progresses, the internal aqueouscontents that include the RNAi agent are delivered into the cell wherethe RNAi agent can specifically bind to a target RNA and can mediateRNAi. In some cases the liposomes are also specifically targeted, e.g.,to direct the RNAi agent to particular cell types.

A liposome containing a RNAi agent can be prepared by a variety ofmethods. In one example, the lipid component of a liposome is dissolvedin a detergent so that micelles are formed with the lipid component. Forexample, the lipid component can be an amphipathic cationic lipid orlipid conjugate. The detergent can have a high critical micelleconcentration and may be nonionic. Exemplary detergents include cholate,CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The RNAiagent preparation is then added to the micelles that include the lipidcomponent. The cationic groups on the lipid interact with the RNAi agentand condense around the RNAi agent to form a liposome. Aftercondensation, the detergent is removed, e.g., by dialysis, to yield aliposomal preparation of RNAi agent.

If necessary a carrier compound that assists in condensation can beadded during the condensation reaction, e.g., by controlled addition.For example, the carrier compound can be a polymer other than a nucleicacid (e.g., spermine or spermidine). pH can also adjusted to favorcondensation.

Methods for producing stable polynucleotide delivery vehicles, whichincorporate a polynucleotide/cationic lipid complex as structuralcomponents of the delivery vehicle, are further described in, e.g., WO96/37194, the entire contents of which are incorporated herein byreference. Liposome formation can also include one or more aspects ofexemplary methods described in Felgner, P. L. et al., (1987) Proc. Natl.Acad. Sci. USA 8:7413-7417; U.S. Pat. Nos. 4,897,355; 5,171,678; Banghamet al., (1965) M Mol. Biol. 23:238; Olson et al., (1979) Biochim.Biophys. Acta 557:9; Szoka et al., (1978) Proc. Natl. Acad. Sci. 75:4194; Mayhew et al., (1984) Biochim. Biophys. Acta 775:169; Kim et al.,(1983) Biochim. Biophys. Acta 728:339; and Fukunaga et al., (1984)Endocrinol. 115:757. Commonly used techniques for preparing lipidaggregates of appropriate size for use as delivery vehicles includesonication and freeze-thaw plus extrusion (see, e.g., Mayer et al.,(1986) Biochim. Biophys. Acta 858:161. Microfluidization can be usedwhen consistently small (50 to 200 nm) and relatively uniform aggregatesare desired (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169. Thesemethods are readily adapted to packaging RNAi agent preparations intoliposomes.

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged nucleicacid molecules to form a stable complex. The positively charged nucleicacid/liposome complex binds to the negatively charged cell surface andis internalized in an endosome. Due to the acidic pH within theendosome, the liposomes are ruptured, releasing their contents into thecell cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun.,147:980-985).

Liposomes, which are pH-sensitive or negatively charged, entrap nucleicacids rather than complex with them. Since both the nucleic acid and thelipid are similarly charged, repulsion rather than complex formationoccurs. Nevertheless, some nucleic acid is entrapped within the aqueousinterior of these liposomes. pH sensitive liposomes have been used todeliver nucleic acids encoding the thymidine kinase gene to cellmonolayers in culture. Expression of the exogenous gene was detected inthe target cells (Zhou et al. (1992) Journal of Controlled Release,19:269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro andin vivo include U.S. Pat. Nos. 5,283,185; 5,171,678; WO 94/00569; WO93/24640; WO 91/16024; Felgner, (1994) J. Biol. Chem. 269:2550; Nabel,(1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther.3:649; Gershon, (1993) Biochem. 32:7143; and Strauss, (1992) EMBO J.11:417.

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporine A into different layers ofthe skin (Hu et al., (1994) S.T.P. Pharma. Sci., 4(6):466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., (1987) FEBSLetters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., (1987), 507:64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., (1988), 85:6949). U.S. Pat. No. 4,837,028 andWO 88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside Gm′ or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

In one embodiment, cationic liposomes are used. Cationic liposomespossess the advantage of being able to fuse to the cell membrane.Non-cationic liposomes, although not able to fuse as efficiently withthe plasma membrane, are taken up by macrophages in vivo and can be usedto deliver RNAi agents to macrophages.

Further advantages of liposomes include: liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated RNAi agents in their internal compartments frommetabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,”Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Importantconsiderations in the preparation of liposome formulations are the lipidsurface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid,N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA)can be used to form small liposomes that interact spontaneously withnucleic acid to form lipid-nucleic acid complexes which are capable offusing with the negatively charged lipids of the cell membranes oftissue culture cells, resulting in delivery of RNAi agent (see, e.g.,Felgner, P. L. et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417,and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use withDNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP)can be used in combination with a phospholipid to form DNA-complexingvesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.)is an effective agent for the delivery of highly anionic nucleic acidsinto living tissue culture cells that comprise positively charged DOTMAliposomes which interact spontaneously with negatively chargedpolynucleotides to form complexes. When enough positively chargedliposomes are used, the net charge on the resulting complexes is alsopositive. Positively charged complexes prepared in this wayspontaneously attach to negatively charged cell surfaces, fuse with theplasma membrane, and efficiently deliver functional nucleic acids into,for example, tissue culture cells. Another commercially availablecationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane(“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMAin that the oleoyl moieties are linked by ester, rather than etherlinkages.

Other reported cationic lipid compounds include those that have beenconjugated to a variety of moieties including, for example,carboxyspermine which has been conjugated to one of two types of lipidsand includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide(“DOGS”) (Transfectam™, Promega, Madison, Wis.) anddipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”)(see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipidwith cholesterol (“DC-Chol”) which has been formulated into liposomes incombination with DOPE (See, Gao, X. and Huang, L., (1991) Biochim.Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugatingpolylysine to DOPE, has been reported to be effective for transfectionin the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta1065:8). For certain cell lines, these liposomes containing conjugatedcationic lipids, are said to exhibit lower toxicity and provide moreefficient transfection than the DOTMA-containing compositions. Othercommercially available cationic lipid products include DMRIE andDMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (LifeTechnology, Inc., Gaithersburg, Md.). Other cationic lipids suitable forthe delivery of oligonucleotides are described in WO 98/39359 and WO96/37194.

Liposomal formulations are particularly suited for topicaladministration, liposomes present several advantages over otherformulations. Such advantages include reduced side effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer RNAi agent into the skin. In some implementations,liposomes are used for delivering RNAi agent to epidermal cells and alsoto enhance the penetration of RNAi agent into dermal tissues, e.g., intoskin. For example, the liposomes can be applied topically. Topicaldelivery of drugs formulated as liposomes to the skin has beendocumented (see, e.g., Weiner et al., (1992) Journal of Drug Targeting,vol. 2, 405-410 and du Plessis et al., (1992) Antiviral Research,18:259-265; Mannino, R. J. and Fould-Fogerite, S., (1998) Biotechniques6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al.(1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. andPapahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. andHuang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver a drug into the dermis of mouse skin. Such formulationswith RNAi agent are useful for treating a dermatological disorder.

Liposomes that include RNAi agents can be made highly deformable. Suchdeformability can enable the liposomes to penetrate through pore thatare smaller than the average radius of the liposome. For example,transfersomes are a type of deformable liposomes. Transferosomes can bemade by adding surface edge activators, usually surfactants, to astandard liposomal composition. Transfersomes that include RNAi agentcan be delivered, for example, subcutaneously by infection in order todeliver RNAi agent to keratinocytes in the skin. In order to crossintact mammalian skin, lipid vesicles must pass through a series of finepores, each with a diameter less than 50 nm, under the influence of asuitable transdermal gradient. In addition, due to the lipid properties,these transferosomes can be self-optimizing (adaptive to the shape ofpores, e.g., in the skin), self-repairing, and can frequently reachtheir targets without fragmenting, and often self-loading.

Other formulations amenable to the present disclosure are described inU.S. provisional application Ser. No. 61/018,616, filed Jan. 2, 2008;61/018,611, filed Jan. 2, 2008; 61/039,748, filed Mar. 26, 2008;61/047,087, filed Apr. 22, 2008 and 61/051,528, filed May 8, 2008. PCTapplication no PCT/US2007/080331, filed Oct. 3, 2007 also describesformulations that are amenable to the present disclosure.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes can be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as thosedescribed herein, particularlay in emulsions (including microemulsions)and liposomes. The most common way of classifying and ranking theproperties of the many different types of surfactants, both natural andsynthetic, is by the use of the hydrophile/lipophile balance (HLB). Thenature of the hydrophilic group (also known as the “head”) provides themost useful means for categorizing the different surfactants used informulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker,Inc., New York, N.Y., 1988, p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides. The use of surfactants in drugproducts, formulations and in emulsions has been reviewed (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

The RNAi agent for use in the methods of the disclosure can also beprovided as micellar formulations. “Micelles” are defined herein as aparticular type of molecular assembly in which amphipathic molecules arearranged in a spherical structure such that all the hydrophobic portionsof the molecules are directed inward, leaving the hydrophilic portionsin contact with the surrounding aqueous phase. The converse arrangementexists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermalmembranes may be prepared by mixing an aqueous solution of the siRNAcomposition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelleforming compounds. Exemplary micelle forming compounds include lecithin,hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid,glycolic acid, lactic acid, chamomile extract, cucumber extract, oleicacid, linoleic acid, linolenic acid, monoolein, monooleates,monolaurates, borage oil, evening of primrose oil, menthol, trihydroxyoxo cholanyl glycine and pharmaceutically acceptable salts thereof,glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethyleneethers and analogues thereof, polidocanol alkyl ethers and analoguesthereof, chenodeoxycholate, deoxycholate, and mixtures thereof. Themicelle forming compounds may be added at the same time or afteraddition of the alkali metal alkyl sulphate. Mixed micelles will formwith substantially any kind of mixing of the ingredients but vigorousmixing in order to provide smaller size micelles.

In one method a first micellar composition is prepared which containsthe siRNA composition and at least the alkali metal alkyl sulphate. Thefirst micellar composition is then mixed with at least three micelleforming compounds to form a mixed micellar composition. In anothermethod, the micellar composition is prepared by mixing the siRNAcomposition, the alkali metal alkyl sulphate and at least one of themicelle forming compounds, followed by addition of the remaining micelleforming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition tostabilize the formulation and protect against bacterial growth.Alternatively, phenol and/or m-cresol may be added with the micelleforming ingredients. An isotonic agent such as glycerin may also beadded after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation canbe put into an aerosol dispenser and the dispenser is charged with apropellant. The propellant, which is under pressure, is in liquid formin the dispenser. The ratios of the ingredients are adjusted so that theaqueous and propellant phases become one, i.e., there is one phase. Ifthere are two phases, it is necessary to shake the dispenser prior todispensing a portion of the contents, e.g., through a metered valve. Thedispensed dose of pharmaceutical agent is propelled from the meteredvalve in a fine spray.

Propellants may include hydrogen-containing chlorofluorocarbons,hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Incertain embodiments, HFA 134a (1,1,1,2 tetrafluoroethane) may be used.

The specific concentrations of the essential ingredients can bedetermined by relatively straightforward experimentation. For absorptionthrough the oral cavities, it is often desirable to increase, e.g., atleast double or triple, the dosage for through injection oradministration through the gastrointestinal tract.

Lipid Particles

RNAi agents, e.g., dsRNAs of in the disclosure may be fully encapsulatedin a lipid formulation, e.g., a LNP, or other nucleic acid-lipidparticle.

As used herein, the term “LNP” refers to a stable nucleic acid-lipidparticle. LNPs typically contain a cationic lipid, a non-cationic lipid,and a lipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). LNPs are extremely useful for systemic applications, as theyexhibit extended circulation lifetimes following intravenous (i.v.)injection and accumulate at distal sites (e.g., sites physicallyseparated from the administration site). LNPs include “pSPLP,” whichinclude an encapsulated condensing agent-nucleic acid complex as setforth in PCT Publication No. WO 00/03683. The particles of the presentdisclosure typically have a mean diameter of about 50 nm to about 150nm, more typically about 60 nm to about 130 nm, more typically about 70nm to about 110 nm, most typically about 70 nm to about 90 nm, and aresubstantially nontoxic. In addition, the nucleic acids when present inthe nucleic acid-lipid particles of the present disclosure are resistantin aqueous solution to degradation with a nuclease. Nucleic acid-lipidparticles and their method of preparation are disclosed in, e.g., U.S.Pat. Nos. 5,976,567; 5,981,501; 6,534,484; 6,586,410; 6,815,432; U.S.Publication No. 2010/0324120 and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1. Ranges intermediate to the above recited ranges are alsocontemplated to be part of the disclosure.

Certain specific LNP formulations for delivery of RNAi agents have beendescribed in the art, including, e.g., “LNP01” formulations asdescribed, e.g., in International Application Publication No. WO2008/042973, which is hereby incorporated by reference.

Additional exemplary lipid-dsRNA formulations are identified in thetable below.

cationic lipid/non-cationic lipid/ cholesterol/PEG-lipid conjugateIonizable/Cationic Lipid Lipid:siRNA ratio SNALP-1l,2-Dilinolenyloxy-N,N- DLinDMA/DPPC/Cholesterol/PEG-cDMAdimethylaminopropane (DLinDMA) (57.1/7.1/34.4/1.4) lipid:siRNA~7:1 2-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2- TechG1/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:1l-yl)ethylazanediyl)didodecan-2-ol (Tech G1) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc-PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-l,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)SNALP (l,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.W02009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference. XTC comprising formulations are described in PCT PublicationNo. WO 2010/088537, the entire contents of which are hereby incorporatedherein by reference. MC3 comprising formulations are described, e.g., inU.S. Publication No. 2010/0324120, filed Jun. 10, 2010, the entirecontents of which are hereby incorporated by reference. ALNY-100comprising formulations are described in PCT Publication No. WO2010/054406, the entire contents of which are hereby incorporated hereinby reference. C12-200 comprising formulations are described in PCTPublication No. WO 2010/129709, the entire contents of which are herebyincorporated herein by reference.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders can be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the disclosure areadministered in conjunction with one or more penetration enhancersurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the disclosure can be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration caninclude sterile aqueous solutions which can also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present disclosure include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions can be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the brain when treatingAPP-associated diseases or disorders.

The pharmaceutical formulations of the present disclosure, which canconveniently be presented in unit dosage form, can be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present disclosure can be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present disclosure can also beformulated as suspensions in aqueous, non-aqueous or mixed media.Aqueous suspensions can further contain substances which increase theviscosity of the suspension including, for example, sodiumcarboxymethylcellulose, sorbitol and/or dextran. The suspension can alsocontain stabilizers.

Additional Formulations

i. Emulsions

The compositions of the present disclosure can be prepared andformulated as emulsions. Emulsions are typically heterogeneous systemsof one liquid dispersed in another in the form of droplets usuallyexceeding 0.1 μm in diameter (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245;Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335;Higuchi et al., in Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa., 1985, p. 301). Emulsions are often biphasic systemscomprising two immiscible liquid phases intimately mixed and dispersedwith each other. In general, emulsions can be of either the water-in-oil(w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finelydivided into and dispersed as minute droplets into a bulk oily phase,the resulting composition is called a water-in-oil (w/o) emulsion.Alternatively, when an oily phase is finely divided into and dispersedas minute droplets into a bulk aqueous phase, the resulting compositionis called an oil-in-water (o/w) emulsion. Emulsions can containadditional components in addition to the dispersed phases, and theactive drug which can be present as a solution in either aqueous phase,oily phase or itself as a separate phase. Pharmaceutical excipients suchas emulsifiers, stabilizers, dyes, and anti-oxidants can also be presentin emulsions as needed. Pharmaceutical emulsions can also be multipleemulsions that are comprised of more than two phases such as, forexample, in the case of oil-in-water-in-oil (o/w/o) andwater-in-oil-in-water (w/o/w) emulsions. Such complex formulations oftenprovide certain advantages that simple binary emulsions do not. Multipleemulsions in which individual oil droplets of an o/w emulsion enclosesmall water droplets constitute a w/o/w emulsion. Likewise a system ofoil droplets enclosed in globules of water stabilized in an oilycontinuous phase provides an o/w/o emulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion can be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatcan be incorporated into either phase of the emulsion. Emulsifiers canbroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich NG., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants can beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that can readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used can be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, LV., Popovich NG., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

ii. Microemulsions

In one embodiment of the present disclosure, the compositions of RNAiagents and nucleic acids are formulated as microemulsions. Amicroemulsion can be defined as a system of water, oil and amphiphilewhich is a single optically isotropic and thermodynamically stableliquid solution (see e.g., Ansel's Pharmaceutical Dosage Forms and DrugDelivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245). Typicallymicroemulsions are systems that are prepared by first dispersing an oilin an aqueous surfactant solution and then adding a sufficient amount ofa fourth component, generally an intermediate chain-length alcohol toform a transparent system. Therefore, microemulsions have also beendescribed as thermodynamically stable, isotropically clear dispersionsof two immiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions can, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase can typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase can include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. Nos. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions can form spontaneously whentheir components are brought together at ambient temperature. This canbe particularly advantageous when formulating thermolabile drugs,peptides or RNAi agents.

Microemulsions have also been effective in the transdermal delivery ofactive components in both cosmetic and pharmaceutical applications. Itis expected that the microemulsion compositions and formulations of thepresent disclosure will facilitate the increased systemic absorption ofRNAi agents and nucleic acids from the gastrointestinal tract, as wellas improve the local cellular uptake of RNAi agents and nucleic acids.

Microemulsions of the present disclosure can also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the RNAi agents and nucleicacids of the present disclosure. Penetration enhancers used in themicroemulsions of the present disclosure can be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

iii. Microparticles

An RNAi agent of the disclosure may be incorporated into a particle,e.g., a microparticle. Microparticles can be produced by spray-drying,but may also be produced by other methods including lyophilization,evaporation, fluid bed drying, vacuum drying, or a combination of thesetechniques.

iv. Penetration Enhancers

In one embodiment, the present disclosure employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly RNAi agents, to the skin of animals. Most drugs are presentin solution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs can cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers can be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants (or “surface-active agents”) are chemical entities which,when dissolved in an aqueous solution, reduce the surface tension of thesolution or the interfacial tension between the aqueous solution andanother liquid, with the result that absorption of RNAi agents throughthe mucosa is enhanced. In addition to bile salts and fatty acids, thesepenetration enhancers include, for example, sodium lauryl sulfate,polyoxyethylene-9-lauryl ether and polyoxyethylene-20-cetyl ether) (seee.g., Malmsten, M. Surfactants and polymers in drug delivery, InformaHealth Care, New York, N.Y., 2002; Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, p. 92); and perfluorochemicalemulsions, such as FC-43. Takahashi et al., J. Pharm. Pharmacol., 1988,40, 252).

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acyl cholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating agents, as used in connection with the present disclosure, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of RNAiagents through the mucosa is enhanced. With regards to their use aspenetration enhancers in the present disclosure, chelating agents havethe added advantage of also serving as DNase inhibitors, as mostcharacterized DNA nucleases require a divalent metal ion for catalysisand are thus inhibited by chelating agents (Jarrett, J. Chromatogr.,1993, 618, 315-339). Suitable chelating agents include but are notlimited to disodium ethylenediaminetetraacetate (EDTA), citric acid,salicylates (e.g., sodium salicylate, 5-methoxysalicylate andhomovanilate), N-acyl derivatives of collagen, laureth-9 and N-aminoacyl derivatives of beta-diketones (enamines)(see e.g., Katdare, A. etal., Excipient development for pharmaceutical, biotechnology, and drugdelivery, CRC Press, Danvers, Mass., 2006; Lee et al., Critical Reviewsin Therapeutic Drug Carrier Systems, 1991, page 92; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al.,J. Control Rel., 1990, 14, 43-51).

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of RNAi agents through the alimentary mucosa (seee.g., Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems,1990, 7, 1-33). This class of penetration enhancers includes, forexample, unsaturated cyclic ureas, 1-alkyl- and1-alkenylazacyclo-alkanone derivatives (Lee et al., Critical Reviews inTherapeutic Drug Carrier Systems, 1991, page 92); and non-steroidalanti-inflammatory agents such as diclofenac sodium, indomethacin andphenylbutazone (Yamashita et al., J. Pharm. Pharmacol., 1987, 39,621-626).

Agents that enhance uptake of RNAi agents at the cellular level can alsobe added to the pharmaceutical and other compositions of the presentdisclosure. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293Fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invitrogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTER™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents can be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

v. Carriers

Certain compositions of the present disclosure also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

vi. Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient can be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent disclosure. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids can includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions can also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

vii. Other Components

The compositions of the present disclosure can additionally containother adjunct components conventionally found in pharmaceuticalcompositions, at their art-established usage levels. Thus, for example,the compositions can contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or cancontain additional materials useful in physically formulating variousdosage forms of the compositions of the present disclosure, such asdyes, flavoring agents, preservatives, antioxidants, opacifiers,thickening agents and stabilizers. However, such materials, when added,should not unduly interfere with the biological activities of thecomponents of the compositions of the present disclosure. Theformulations can be sterilized and, if desired, mixed with auxiliaryagents, e.g., lubricants, preservatives, stabilizers, wetting agents,emulsifiers, salts for influencing osmotic pressure, buffers, colorings,flavorings and/or aromatic substances and the like which do notdeleteriously interact with the nucleic acid(s) of the formulation.

Aqueous suspensions can contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension can also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in thedisclosure include (a) one or more RNAi agents and (b) one or moreagents which function by a non-RNAi mechanism and which are useful intreating an APP-associated disorder. Examples of such agents include,but are not limited to an anti-inflammatory agent, anti-steatosis agent,anti-viral, and/or anti-fibrosis agent, or other agent included to treatAD (including EOFAD) and/or CAA in a subject.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD₅₀ (the dose lethal to 50% of thepopulation) and the ED₅₀ (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD₅₀/ED₅₀.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured herein in the disclosure lies generally within arange of circulating concentrations that include the ED₅₀ with little orno toxicity. The dosage can vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the disclosure, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose can be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC₅₀ (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma can be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the RNAi agentsfeatured in the disclosure can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby APP expression. In any event, the administering physician can adjustthe amount and timing of RNAi agent administration on the basis ofresults observed using standard measures of efficacy known in the art ordescribed herein.

VIII. Kits

In certain aspects, the instant disclosure provides kits that include asuitable container containing a pharmaceutical formulation of a siRNAcompound, e.g., a double-stranded siRNA compound, or ssiRNA compound,(e.g., a precursor, e.g., a larger siRNA compound which can be processedinto a ssiRNA compound, or a DNA which encodes an siRNA compound, e.g.,a double-stranded siRNA compound, or ssiRNA compound, or precursorthereof). In certain embodiments the individual components of thepharmaceutical formulation may be provided in one container.Alternatively, it may be desirable to provide the components of thepharmaceutical formulation separately in two or more containers, e.g.,one container for a siRNA compound preparation, and at least another fora carrier compound. The kit may be packaged in a number of differentconfigurations such as one or more containers in a single box. Thedifferent components can be combined, e.g., according to instructionsprovided with the kit. The components can be combined according to amethod described herein, e.g., to prepare and administer apharmaceutical composition. The kit can also include a delivery device.

IX. Methods for Inhibiting APP Expression

The present disclosure also provides methods of inhibiting expression ofan APP gene in a cell. The methods include contacting a cell with anRNAi agent, e.g., double stranded RNAi agent, in an amount effective toinhibit expression of APP in the cell, thereby inhibiting expression ofAPP in the cell. In certain embodiments of the disclosure, APP isinhibited preferentially in CNS (e.g., brain) cells.

Contacting of a cell with a RNAi agent, e.g., a double stranded RNAiagent, may be done in vitro or in vivo. Contacting a cell in vivo withthe RNAi agent includes contacting a cell or group of cells within asubject, e.g., a human subject, with the RNAi agent. Combinations of invitro and in vivo methods of contacting a cell are also possible.

Contacting a cell may be direct or indirect, as discussed above.Furthermore, contacting a cell may be accomplished via a targetingligand, including any ligand described herein or known in the art. Insome embodiments, the targeting ligand is a carbohydrate moiety, e.g., aC16 ligand, or any other ligand that directs the RNAi agent to a site ofinterest.

The term “inhibiting,” as used herein, is used interchangeably with“reducing,” “silencing,” “downregulating,” “suppressing” and othersimilar terms, and includes any level of inhibition. In certainembodiments, a level of inhibition, e.g., for a RNAi agent of theinstant disclosure, can be assessed in cell culture conditions, e.g.,wherein cells in cell culture are transfected viaLipofectamine™-mediated transfection at a concentration in the vicinityof a cell of 10 nM or less, 1 nM or less, etc. Knockdown of a given RNAiagent can be determined via comparison of pre-treated levels in cellculture versus post-treated levels in cell culture, optionally alsocomparing against cells treated in parallel with a scrambled or otherform of control RNAi agent. Knockdown in cell culture of, e.g., at least10% or more, at least 20% or more, etc. can thereby be identified asindicative of “inhibiting” and/or “reducing”, “downregulating” or“suppressing”, etc. having occurred. It is expressly contemplated thatassessment of targeted mRNA and/or encoded protein levels (and thereforean extent of “inhibiting”, etc. caused by a RNAi agent of thedisclosure) can also be assessed in in vivo systems for the RNAi agentsof the instant disclosure, under properly controlled conditions asdescribed in the art.

The phrase “inhibiting expression of an APP,” as used herein, includesinhibition of expression of any APP gene (such as, e.g., a mouse APPgene, a rat APP gene, a monkey APP gene, or a human APP gene) as well asvariants or mutants of an APP gene that encode an APP protein. Thus, theAPP gene may be a wild-type APP gene, a mutant APP gene, or a transgenicAPP gene in the context of a genetically manipulated cell, group ofcells, or organism.

“Inhibiting expression of an APP gene” includes any level of inhibitionof an APP gene, e.g., at least partial suppression of the expression ofan APP gene, such as an inhibition by at least about 20%. In certainembodiments, inhibition is by at least about 25%, at least about 30%, atleast about 35%, at least about 40%, at least about 45%, at least about50%, at least about 55%, at least about 60%, at least about 65%, atleast about 70%, at least about 75%, at least about 80%, at least about85%, at least about 90%, at least about 91%, at least about 92%, atleast about 93%, at least about 94%, at least about 95%, at least about96%, at least about 97%, at least about 98%, or at least about 99%.

The expression of an APP gene may be assessed based on the level of anyvariable associated with APP gene expression, e.g., APP mRNA level orAPP protein level (including APP cleavage products). The expression ofan APP may also be assessed indirectly based on the levels ofAPP-associated biomarkers as described herein.

Inhibition may be assessed by a decrease in an absolute or relativelevel of one or more of these variables compared with a control level.The control level may be any type of control level that is utilized inthe art, e.g., a pre-dose baseline level, or a level determined from asimilar subject, cell, or sample that is untreated or treated with acontrol (such as, e.g., buffer only control or inactive agent control).

In certain embodiments, surrogate markers can be used to detectinhibition of APP. For example, effective prevention or treatment of anAPP-associated disorder, e.g., a CNS disorder such as EOFAD, CAA orother disorder, as demonstrated by acceptable diagnostic and monitoringcriteria with an agent to reduce APP expression can be understood todemonstrate a clinically relevant reduction in APP.

In some embodiments of the methods of the disclosure, expression of anAPP gene is inhibited by at least 20%, a 25%, 30%, 35%, 40%, 45%, 50%,55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or to below the level ofdetection of the assay. In certain embodiments, the methods include aclinically relevant inhibition of expression of APP, e.g. asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of APP.

Inhibition of the expression of an APP gene may be manifested by areduction of the amount of mRNA expressed by a first cell or group ofcells (such cells may be present, for example, in a sample derived froma subject) in which an APP gene is transcribed and which has or havebeen treated (e.g., by contacting the cell or cells with a RNAi agent ofthe disclosure, or by administering a RNAi agent of the disclosure to asubject in which the cells are or were present) such that the expressionof an APP gene is inhibited, as compared to a second cell or group ofcells substantially identical to the first cell or group of cells butwhich has not or have not been so treated (control cell(s) not treatedwith a RNAi agent or not treated with a RNAi agent targeted to the geneof interest). The degree of inhibition may be expressed in terms of:

${\frac{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right) - \left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{treated}\mspace{14mu}{cells}} \right)}{\left( {{mRNA}\mspace{14mu}{in}\mspace{14mu}{control}\mspace{14mu}{cells}} \right)} \cdot 100}\%$

In other embodiments, inhibition of the expression of an APP gene may beassessed in terms of a reduction of a parameter that is functionallylinked to APP gene expression, e.g., APP protein expression, formationand/or levels of APP cleavage products, or APP signaling pathways. APPgene silencing may be determined in any cell expressing APP, eitherendogenous or heterologous from an expression construct, and by anyassay known in the art.

Inhibition of the expression of an APP protein may be manifested by areduction in the level of the APP protein that is expressed by a cell orgroup of cells (e.g., the level of protein expressed in a sample derivedfrom a subject). As explained above, for the assessment of mRNAsuppression, the inhibition of protein expression levels in a treatedcell or group of cells may similarly be expressed as a percentage of thelevel of protein in a control cell or group of cells.

A control cell or group of cells that may be used to assess theinhibition of the expression of an APP gene includes a cell or group ofcells that has not yet been contacted with a RNAi agent of thedisclosure. For example, the control cell or group of cells may bederived from an individual subject (e.g., a human or animal subject)prior to treatment of the subject with an RNAi agent.

The level of APP mRNA that is expressed by a cell or group of cells maybe determined using any method known in the art for assessing mRNAexpression. In one embodiment, the level of expression of APP in asample is determined by detecting a transcribed polynucleotide, orportion thereof, e.g., mRNA of the APP gene. RNA may be extracted fromcells using RNA extraction techniques including, for example, using acidphenol/guanidine isothiocyanate extraction (RNAzol B; Biogenesis),RNeasy™ RNA preparation kits (Qiagen®) or PAXgene (PreAnalytix,Switzerland). Typical assay formats utilizing ribonucleic acidhybridization include nuclear run-on assays, RT-PCR, RNase protectionassays, northern blotting, in situ hybridization, and microarrayanalysis. Circulating APP mRNA may be detected using methods thedescribed in PCT Publication WO2012/177906, the entire contents of whichare hereby incorporated herein by reference.

In some embodiments, the level of expression of APP is determined usinga nucleic acid probe. The term “probe”, as used herein, refers to anymolecule that is capable of selectively binding to a specific APP.Probes can be synthesized by one of skill in the art, or derived fromappropriate biological preparations. Probes may be specifically designedto be labeled. Examples of molecules that can be utilized as probesinclude, but are not limited to, RNA, DNA, proteins, antibodies, andorganic molecules.

Isolated mRNA can be used in hybridization or amplification assays thatinclude, but are not limited to, Southern or northern analyses,polymerase chain reaction (PCR) analyses and probe arrays. One methodfor the determination of mRNA levels involves contacting the isolatedmRNA with a nucleic acid molecule (probe) that can hybridize to APPmRNA. In one embodiment, the mRNA is immobilized on a solid surface andcontacted with a probe, for example by running the isolated mRNA on anagarose gel and transferring the mRNA from the gel to a membrane, suchas nitrocellulose. In an alternative embodiment, the probe(s) areimmobilized on a solid surface and the mRNA is contacted with theprobe(s), for example, in an Affymetrix gene chip array. A skilledartisan can readily adapt known mRNA detection methods for use indetermining the level of APP mRNA.

An alternative method for determining the level of expression of APP ina sample involves the process of nucleic acid amplification and/orreverse transcriptase (to prepare cDNA) of for example mRNA in thesample, e.g., by RT-PCR (the experimental embodiment set forth inMullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany(1991) Proc. Natl. Acad. Sci. USA 88:189-193), self sustained sequencereplication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA87:1874-1878), transcriptional amplification system (Kwoh et al. (1989)Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-Beta Replicase (Lizardi etal. (1988) Bio/Technology 6:1197), rolling circle replication (Lizardiet al., U.S. Pat. No. 5,854,033) or any other nucleic acid amplificationmethod, followed by the detection of the amplified molecules usingtechniques well known to those of skill in the art. These detectionschemes are especially useful for the detection of nucleic acidmolecules if such molecules are present in very low numbers. Inparticular aspects of the disclosure, the level of expression of APP isdetermined by quantitative fluorogenic RT-PCR (i.e., the TaqMan™System), by a Dual-Glo® Luciferase assay, or by other art-recognizedmethod for measurement of APP expression and/or mRNA level.

The expression levels of APP mRNA may be monitored using a membrane blot(such as used in hybridization analysis such as northern, Southern, dot,and the like), or microwells, sample tubes, gels, beads or fibers (orany solid support comprising bound nucleic acids). See U.S. Pat. Nos.5,770,722, 5,874,219, 5,744,305, 5,677,195 and 5,445,934, which areincorporated herein by reference. The determination of APP expressionlevel may also comprise using nucleic acid probes in solution.

In some embodiments, the level of mRNA expression is assessed usingbranched DNA (bDNA) assays or real time PCR (qPCR). The use of this PCRmethod is described and exemplified in the Examples presented herein.Such methods can also be used for the detection of APP nucleic acids,SREBP nucleic acids or PNPLA3 nucleic acids.

The level of APP protein expression may be determined using any methodknown in the art for the measurement of protein levels. Such methodsinclude, for example, electrophoresis, capillary electrophoresis, highperformance liquid chromatography (HPLC), thin layer chromatography(TLC), hyperdiffusion chromatography, fluid or gel precipitin reactions,absorption spectroscopy, a colorimetric assays, spectrophotometricassays, flow cytometry, immunodiffusion (single or double),immunoelectrophoresis, western blotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assays (ELISAs), immunofluorescent assays,electrochemiluminescence assays, and the like. Such assays can also beused for the detection of proteins indicative of the presence orreplication of APP proteins, APP cleavage products, or other proteinsassociated with APP, e.g., PSEN1, PSEN2, etc.

In some embodiments, the efficacy of the methods of the disclosure inthe treatment of an APP-related disease is assessed by a decrease in APPmRNA level (e.g, by assessment of a CSF sample for Aβ levels, by brainbiopsy, or otherwise).

In some embodiments of the methods of the disclosure, the RNAi agent isadministered to a subject such that the RNAi agent is delivered to aspecific site within the subject. The inhibition of expression of APPmay be assessed using measurements of the level or change in the levelof APP mRNA or APP protein in a sample derived from a specific sitewithin the subject, e.g., CNS cells. In certain embodiments, the methodsinclude a clinically relevant inhibition of expression of APP, e.g. asdemonstrated by a clinically relevant outcome after treatment of asubject with an agent to reduce the expression of APP.

As used herein, the terms detecting or determining a level of an analyteare understood to mean performing the steps to determine if a material,e.g., protein, RNA, is present. As used herein, methods of detecting ordetermining include detection or determination of an analyte level thatis below the level of detection for the method used.

X. Methods of Treating or Preventing APP-Associated Diseases

The present disclosure also provides methods of using a RNAi agent ofthe disclosure and/or a composition containing a RNAi agent of thedisclosure to reduce and/or inhibit APP expression in a cell. Themethods include contacting the cell with a dsRNA of the disclosure andmaintaining the cell for a time sufficient to obtain degradation of themRNA transcript of an APP gene, thereby inhibiting expression of the APPgene in the cell. Reduction in gene expression can be assessed by anymethods known in the art. For example, a reduction in the expression ofAPP may be determined by determining the mRNA expression level of APPusing methods routine to one of ordinary skill in the art, e.g.,Northern blotting, qRT-PCR; by determining the protein level of APPusing methods routine to one of ordinary skill in the art, such asWestern blotting, immunological techniques. A reduction in theexpression of APP may also be assessed indirectly by measuring adecrease in the levels of a soluble cleavage product of APP, e.g., adecrease in the level of soluble APPα, APPβ and/or a soluble AP peptide,optionally in a CSF sample of a subject.

In the methods of the disclosure the cell may be contacted in vitro orin vivo, i.e., the cell may be within a subject.

A cell suitable for treatment using the methods of the disclosure may beany cell that expresses an APP gene. A cell suitable for use in themethods of the disclosure may be a mammalian cell, e.g., a primate cell(such as a human cell or a non-human primate cell, e.g., a monkey cellor a chimpanzee cell), a non-primate cell (such as a cow cell, a pigcell, a camel cell, a llama cell, a horse cell, a goat cell, a rabbitcell, a sheep cell, a hamster, a guinea pig cell, a cat cell, a dogcell, a rat cell, a mouse cell, a lion cell, a tiger cell, a bear cell,or a buffalo cell), a bird cell (e.g., a duck cell or a goose cell), ora whale cell. In one embodiment, the cell is a human cell, e.g., a humanCNS cell.

APP expression is inhibited in the cell by at least about 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 45, 46,47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, orabout 100%. In preferred embodiments, APP expression is inhibited by atleast 20%.

The in vivo methods of the disclosure may include administering to asubject a composition containing a RNAi agent, where the RNAi agentincludes a nucleotide sequence that is complementary to at least a partof an RNA transcript of the APP gene of the mammal to be treated. Whenthe organism to be treated is a mammal such as a human, the compositioncan be administered by any means known in the art including, but notlimited to oral, intraperitoneal, or parenteral routes, includingintracranial (e.g., intraventricular, intraparenchymal and intrathecal),intravenous, intramuscular, intravitreal, subcutaneous, transdermal,airway (aerosol), nasal, rectal, and topical (including buccal andsublingual) administration. In certain embodiments, the compositions areadministered by intravenous infusion or injection. In certainembodiments, the compositions are administered by subcutaneousinjection.

In some embodiments, the administration is via a depot injection. Adepot injection may release the RNAi agent in a consistent way over aprolonged time period. Thus, a depot injection may reduce the frequencyof dosing needed to obtain a desired effect, e.g., a desired inhibitionof APP, or a therapeutic or prophylactic effect. A depot injection mayalso provide more consistent serum concentrations. Depot injections mayinclude subcutaneous injections or intramuscular injections. Inpreferred embodiments, the depot injection is a subcutaneous injection.

In some embodiments, the administration is via a pump. The pump may bean external pump or a surgically implanted pump. In certain embodiments,the pump is a subcutaneously implanted osmotic pump. In otherembodiments, the pump is an infusion pump. An infusion pump may be usedfor intravenous, subcutaneous, arterial, or epidural infusions. Inpreferred embodiments, the infusion pump is a subcutaneous infusionpump. In other embodiments, the pump is a surgically implanted pump thatdelivers the RNAi agent to the CNS.

The mode of administration may be chosen based upon whether local orsystemic treatment is desired and based upon the area to be treated. Theroute and site of administration may be chosen to enhance targeting.

In one aspect, the present disclosure also provides methods forinhibiting the expression of an APP gene in a mammal. The methodsinclude administering to the mammal a composition comprising a dsRNAthat targets an APP gene in a cell of the mammal and maintaining themammal for a time sufficient to obtain degradation of the mRNAtranscript of the APP gene, thereby inhibiting expression of the APPgene in the cell. Reduction in gene expression can be assessed by anymethods known it the art and by methods, e.g. qRT-PCR, described herein.Reduction in protein production can be assessed by any methods known itthe art and by methods, e.g. ELISA, described herein. In one embodiment,a CNS biopsy sample or a cerebrospinal fluid (CSF) sample serves as thetissue material for monitoring the reduction in APP gene and/or proteinexpression (or of a proxy therefore, as described herein or as known inthe art).

The present disclosure further provides methods of treatment of asubject in need thereof. The treatment methods of the disclosure includeadministering a RNAi agent of the disclosure to a subject, e.g., asubject that would benefit from a reduction and/or inhibition of APPexpression, in a therapeutically effective amount of a RNAi agenttargeting an APP gene or a pharmaceutical composition comprising a RNAiagent targeting an APP gene.

The present disclosure also provides methods of decreasing Aβ40 and/orAβ42 levels in a subject. The methods include administering a RNAi agentof the disclosure to a subject, e.g., a subject that would benefit froma reduction and/or inhibition of APP expression, in a therapeuticallyeffective amount of a RNAi agent targeting an APP gene or apharmaceutical composition comprising a RNAi agent targeting an APPgene.

In addition, the present disclosure provides methods of preventing,treating and/or inhibiting the progression of an APP-associated diseaseor disorder (e.g., CAA and/or AD, optionally EOFAD) in a subject, suchas the progression of an APP-associated disease or disorder toneurodegeneration, increased amyloid plaque formation and/or cognitivedecline in a subject having an APP-associated disease or disorder or asubject at risk of developing an APP-associated disease or disorder. Themethods include administering to the subject a therapeutically effectiveamount of any of the dsRNAs or the pharmaceutical composition providedherein, thereby preventing, treating and/or inhibiting the progressionof an APP-associated disease or disorder in the subject.

A RNAi agent of the disclosure may be administered as a “free RNAiagent.” A free RNAi agent is administered in the absence of apharmaceutical composition. The naked RNAi agent may be in a suitablebuffer solution. The buffer solution may comprise acetate, citrate,prolamine, carbonate, or phosphate, or any combination thereof. In oneembodiment, the buffer solution is phosphate buffered saline (PBS). ThepH and osmolarity of the buffer solution containing the RNAi agent canbe adjusted such that it is suitable for administering to a subject.

Alternatively, a RNAi agent of the disclosure may be administered as apharmaceutical composition, such as a dsRNA liposomal formulation.

Subjects that would benefit from a reduction and/or inhibition of APPgene expression are those having an APP-associated disorder. The term“APP-associated disease” includes a disease, disorder or condition thatwould benefit from a decrease in APP gene expression, replication, orprotein activity. Non-limiting examples of APP-associated diseasesinclude, for example, CAA (including hCAA and sporadic CAA) and AD(including EOFAD, sporadic and/or late onset AD, optionally with CAA).

The disclosure further provides methods for the use of a RNAi agent or apharmaceutical composition thereof, e.g., for treating a subject thatwould benefit from reduction and/or inhibition of APP expression, e.g.,a subject having an APP-associated disorder, in combination with otherpharmaceuticals and/or other therapeutic methods, e.g., with knownpharmaceuticals and/or known therapeutic methods, such as, for example,those which are currently employed for treating these disorders. Forexample, in certain embodiments, a RNAi agent targeting APP isadministered in combination with, e.g., an agent useful in treating anAPP-associated disorder as described elsewhere herein or as otherwiseknown in the art. For example, additional agents suitable for treating asubject that would benefit from reduction in APP expression, e.g., asubject having an APP-associated disorder, may include agents currentlyused to treat symptoms of AD. Non-limiting examples of such agents mayinclude cholinesterase inhibitors (such as donepezil, rivastigmate, andgalantamine), memantine, BACEli, immunotherapies, and secretaseinhibitors. The RNAi agent and additional therapeutic agents may beadministered at the same time and/or in the same combination, e.g.,intrathecally, or the additional therapeutic agent can be administeredas part of a separate composition or at separate times and/or by anothermethod known in the art or described herein.

In one embodiment, the method includes administering a compositionfeatured herein such that expression of the target APP gene isdecreased, such as for about 1, 2, 3, 4, 5, 6, 7, 8, 12, 16, 18, 24hours, 28, 32, or about 36 hours. In one embodiment, expression of thetarget APP gene is decreased for an extended duration, e.g., at leastabout two, three, four days or more, e.g., about one week, two weeks,three weeks, or four weeks or longer.

Preferably, the RNAi agents useful for the methods and compositionsfeatured herein specifically target RNAs (primary or processed) of thetarget APP gene. Compositions and methods for inhibiting the expressionof these genes using RNAi agents can be prepared and performed asdescribed herein.

Administration of the dsRNA according to the methods of the disclosuremay result in a reduction of the severity, signs, symptoms, and/ormarkers of such diseases or disorders in a patient with anAPP-associated disorder. By “reduction” in this context is meant astatistically significant decrease in such level. The reduction can be,for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or about 100%.

Efficacy of treatment or prevention of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Forexample, efficacy of treatment of an APP-associated disorder may beassessed, for example, by periodic monitoring of a subject's cognition,CSF AP levels, etc. Comparisons of the later readings with the initialreadings provide a physician an indication of whether the treatment iseffective. It is well within the ability of one skilled in the art tomonitor efficacy of treatment or prevention by measuring any one of suchparameters, or any combination of parameters. In connection with theadministration of a RNAi agent targeting APP or pharmaceuticalcomposition thereof, “effective against” an APP-associated disorderindicates that administration in a clinically appropriate manner resultsin a beneficial effect for at least a statistically significant fractionof patients, such as an improvement of symptoms, a cure, a reduction indisease, extension of life, improvement in quality of life, or othereffect generally recognized as positive by medical doctors familiar withtreating APP-associated disorders and the related causes.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given RNAi agent drug or formulation of thatdrug can also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

Alternatively, the efficacy can be measured by a reduction in theseverity of disease as determined by one skilled in the art of diagnosisbased on a clinically accepted disease severity grading scale, as butone example mental ability tests for dementia. Any positive changeresulting in e.g., lessening of severity of disease measured using theappropriate scale, represents adequate treatment using a RNAi agent orRNAi agent formulation as described herein.

Subjects can be administered a therapeutic amount of dsRNA, such asabout 0.01 mg/kg to about 200 mg/kg.

The RNAi agent can be administered intrathecally, via intravitrealinjection and/or by intravenous infusion over a period of time, on aregular basis. In certain embodiments, after an initial treatmentregimen, the treatments can be administered on a less frequent basis.Administration of the RNAi agent can reduce APP levels, e.g., in a cell,tissue, blood, CSF sample or other compartment of the patient by atleast about 5%, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38,39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 39, 50, 51, 52, 53, 54, 55, 56,57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92,93, 94, 95, 96, 97, 98, or at least about 99% or more. In a preferredembodiment, administration of the RNAi agent can reduce APP levels,e.g., in a cell, tissue, blood, CSF sample or other compartment of thepatient by at least 20%.

Before administration of a full dose of the RNAi agent, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction. In anotherexample, the patient can be monitored for unwanted immunostimulatoryeffects, such as increased cytokine (e.g., TNF-alpha or INF-alpha)levels.

Alternatively, the RNAi agent can be administered subcutaneously, i.e.,by subcutaneous injection. One or more injections may be used to deliverthe desired, e.g., monthly dose of RNAi agent to a subject. Theinjections may be repeated over a period of time. The administration maybe repeated on a regular basis. In certain embodiments, after an initialtreatment regimen, the treatments can be administered on a less frequentbasis. A repeat-dose regimine may include administration of atherapeutic amount of RNAi agent on a regular basis, such as monthly orextending to once a year or once every 2, 3, 4 and/or 5 years. Incertain embodiments, the RNAi agent is administered about once per monthto about once per quarter (i.e., about once every three months).

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the RNAi agents and methods featured in theinvention, suitable methods and materials are described below. Allpublications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, the materials, methods, and examples areillustrative only and not intended to be limiting.

EXAMPLES Example 1. RNAi Agent Design, Synthesis, Selection, and InVitro Evaluation

This Example describes methods for the design, synthesis, selection, andin vitro evaluation of APP RNAi agents.

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Bioinformatics

A set of siRNA agents targeting the human amyloid beta precursor proteingene (APP; human NCBI refseq NM_201414; NCBI GeneID: 351; SEQ ID NO: 1),as well as the toxicology-species APP ortholog from Macaca fascicularis(cynomolgus monkey: XM_005548883.2; SEQ ID NO: 12) was designed usingcustom R and Python scripts. All the siRNA designs have a perfect matchto the human APP transcript and a subset either perfect or near-perfectmatches to the cynomolgus ortholog. The human NM_201414 REFSEQ mRNA,version 2, has a length of 3423 bases. The rationale and method for theset of siRNA designs is as follows: the predicted efficacy for everypotential 23mer siRNA from position 10 through the end was determinedwith a random forest model derived from the direct measure of mRNAknockdown from several thousand distinct siRNA designs targeting adiverse set of vertebrate genes. For each strand of the siRNA, a customPython script was used in a brute force search to measure the number andpositions of mismatches between the siRNA and all potential alignmentsin the human transcriptome. Extra weight was given to mismatches in theseed region, defined here as positions 2-9 of the antisenseoligonucleotide, as well the cleavage site of the siRNA, defined here aspositions 10-11 of the antisense oligonucleotide. The relative weight ofthe mismatches was 2.8, 1.2, 1 for seed mismatches, cleavage site, andother positions up through antisense position 19. Mismatches in thefirst position were ignored. A specificity score was calculated for eachstrand by summing the value of each weighted mismatch. Preference wasgiven to siRNAs whose antisense score in human and monkey was ≥3 with apredicted efficacy of ≥50% knockdown (161 sequences), or with anantisense score ≥2 and ≥60% predicted knockdown (118 sequences).

A second set of siRNAs targeting the toxicology-species Mus musculus(mouse) amyloid beta precursor protein (App, an ortholog of the humanAPP; mouse NCBI refseq NM_001198823; NCBI GeneID: 11820; SEQ ID NO: 13)as well as the Rattus norvegicus (rat) App ortholog: NM_019288.2 (SEQ IDNO: 14) was designed using custom R and Python scripts. All the siRNAdesigns possessed a perfect match to the mouse App transcript and asubset possessed either perfect or near-perfect matches to the ratortholog. The mouse NM_001198823 REFSEQ mRNA, version 1, has a length of3377 bases. The same selection process was used as stated above forhuman sequences, but with the following selection criteria applied:Preference was given to siRNAs whose antisense score in mouse and ratwas ≥2.8 with a predicted efficacy of ≥50% knockdown (85 sequences), orwith an antisense score ≥2 and ≥61% predicted knockdown (8 sequences).

Synthesis of APP Sequences

Synthesis of APP Single Strands and Duplexes

All oligonucleotides were prepared on a MerMade 192 synthesizer on a 1μmole scale using universal or custom supports. All phosphoramiditeswere used at a concentration 100 mM in 100% Acetonitrile or 9:1Acetonitrile:DMF with a standard protocol for 2-cyanoethylphosphoramidites, except that the coupling time was extended to 400seconds. Oxidation of the newly formed linkages was achieved using asolution of 50 mM I₂ in 9:1 Acetonitrile:Water to create phosphatelinkages and 100 mM DDTT in 9:1 Pyridine:Acetonitrile to createphosphorothioate linkages. After the trityl-off synthesis, columns wereincubated with 150 μL of 40% aqueous Methylamine for 45 minutes and thesolution drained via vacuum into a 96-well plate. After repeating theincubation and draining with a fresh portion of aqueous Methylamine, theplate containing crude oligonucleotide solution was sealed and shaken atroom temperature for an additional 60 minutes to completely remove allprotecting groups. Precipitation of the crude oligonucleotides wasaccomplished via the addition of 1.2 mL of 9:1 Acetonitrile:EtOH to eachwell followed by incubation at −20° C. overnight. The plate was thencentrifuged at 3000 RPM for 45 minutes, the supernatant removed fromeach well, and the pellets resuspended in 950 μL of 20 mM aqueous NaOAc.Each crude solution was finally desalted over a GE Hi-Trap DesaltingColumn (Sephadex G25 Superfine) using water to elute the finaloligonucleotide products. All identities and purities were confirmedusing ESI-MS and IEX HPLC, respectively.

Annealing of APP single strands was performed on a Tecan liquid handlingrobot. Sense and antisense single strands were combined in an equimolarratio in 96 well plates and buffered with 10×PBS to provide a finalduplex concentration of 10 μM in 1×PBS. After combining thecomplementary single strands, the 96 well plate was sealed tightly andheated in an oven at 100° C. for 40 minutes and allowed to come slowlyto room temperature over a period of 2-3 hours and subsequently useddirectly for in vitro screening assays at the appropriateconcentrations.

A detailed list of the modified APP sense and antisense strand sequencesis shown in Tables 2A, 2B, 3, 5A, 5B, 6, 9, 10-15, 16A, 16B, and 26 anda detailed list of the unmodified APP sense and antisense strandsequences is shown in Tables 3, 6, 11, 13, 15, and 26.

In Vitro Primary Mouse, Primary Cynomolgus Hepatocytes, be(2)C andNeuron2A Screening:

Cell Culture and Transfections:

Human Be(2)C (ATCC), mouse Neuro2A (ATCC), Primary Mouse Hepatocytes(BioreclamationIVT) and Primary cyno hepatocytes (BioreclamationIVT)were transfected by adding 4.9 μl of Opti-MEM plus 0.1 μl of RNAiMAX perwell (Invitrogen, Carlsbad Calif. cat #13778-150) to 5 μl of siRNAduplexes per well, with 4 replicates of each siRNA duplex, into a384-well plate, and incubated at room temperature for 15 minutes. 40 μlof media containing ˜5×10³ cells were then added to the siRNA mixture.Cells were incubated for 24 hours prior to RNA purification. Multi-doseexperiments were performed at 10 nM and 0.1 nM.

Total RNA Isolation Using DYNABEADS mRNA Isolation Kit (Invitrogen, Part#: 610-12):

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 70 μl ofLysis/Binding Buffer and 10 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand supernatant removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

10 μl of a master mix containing 1 μl 10× Buffer, 0.4 μl 25× dNTPs, 1 μl10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H₂O per reaction was added to RNA isolated above. Plateswere sealed, mixed, and incubated on an electromagnetic shaker for 10minutes at room temperature, followed by 2 h 37° C.

Real Time PCR:

Two μl of cDNA were added to a master mix containing 0.5 μl of humanGAPDH TaqMan Probe (4326317E), and 0.5 μl APP human probe(Hs00169098_m1) and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) per well in a 384 well plates (Roche cat #04887301001). Or2 μl of cDNA were added to a master mix containing 0.5 μl of mouse GAPDHTaqMan Probe (4352339E), and 0.5 μl APP mouse probe (Mm01344172_m1) and5 μl Lightcycler 480 probe master mix (Roche Cat #04887301001) per wellin a 384 well plates (Roche cat #04887301001). Or 2 μl of cDNA wereadded to a master mix containing 0.5 μl of Cyno GAPDH TaqMan Probe(forward primer: 5′-GCATCCTGGGCTACACTGA-3′, reverse primer:5′-TGGGTGTCGCTGTTGAAGTC-3′, probe: 5′HEX-CCAGGTGGTCTCCTCC-3′BHQ-1) and0.5 μl APP cynomolgus probe (Mf01552291_m1) and 5 μl Lightcycler 480probe master mix (Roche Cat #04887301001) per well in a 384 well plates(Roche cat #04887301001). Real time PCR was done in a LightCycler480Real Time PCR system (Roche). Each duplex was tested at least two timesand data were normalized to cells transfected with a non-targetingcontrol siRNA.

To calculate relative fold change, real time data were analyzed usingthe ΔΔCt method and normalized to assays performed with cellstransfected with a non-targeting control siRNA. The results from theassays are shown in Tables 4 and 7.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) AAdenosine-3′-phosphate Agn (S)-glycol-adenosine Ahd 2′-O-hexadecyladenosine-3′-phosphate Af 2′-fluoroadenosine-3′-phosphate Afs2′-fluoroadenosine-3′-phosphorothioate As adenosine-3′-phosphorothioateC cytidine-3′-phosphate Cgn (S)-glycol-cytidine Chd 2′-O-hexadecylcytidine-3′-phosphate Cf 2′-fluorocytidine-3′-phosphate Cfs2′-fluorocytidine-3′-phosphorothioate Cs cytidine-3′-phosphorothioate Gguanosine-3′-phosphate Ggn (S)-glycol-guanosine Ghd 2′-O-hexadecylguanosine-3′-phosphate Gf 2′-fluoroguanosine-3′-phosphate Gfs2′-fluoroguanosine-3′-phosphorothioate Gs guanosine-3′-phosphorothioateT 5′-methyluridine-3′-phosphate Tgn (S)-glycol-5′-methyluridine Tf2′-fluoro-5-methyluridine-3′-phosphate Tfs2′-fluoro-5-methyluridine-3′-phosphorothioate Ts5-methyluridine-3′-phosphorothioate U Uridine-3′-phosphate Uhd2′-O-hexadecyl uridine-3′-phosphate Uf 2′-fluorouridine-3′-phosphate Ufs2′-fluorouridine -3′-phosphorothioate Us uridine -3′-phosphorothioate Nany nucleotide (G, A, C, T or U) a 2′-O-methyladenosine-3′-phosphate as2′-O-methyladenosine-3′- phosphorothioate c2′-O-methylcytidine-3′-phosphate cs 2′-O-methylcytidine-3′-phosphorothioate g 2′-O-methylguanosine-3′-phosphate gs2′-O-methylguanosine-3′- phosphorothioate t2′-O-methyl-5-methyluridine-3′-phosphate ts2′-O-methyl-5-methyluridine-3′-phosphorothioate u2′-O-methyluridine-3′-phosphate us2′-O-methyluridine-3′-phosphorothioate s phosphorothioate linkage L96N-[tris(GalNAc-alkyl)-amidodecanoyl)]-4-hydroxyprolinolHyp-(GalNAc-alkyl)3 dT 2′-deoxythymidine-3′-phosphate dC2′-deoxycytidine-3′-phosphate P Phosphate VP Vinyl-phosphonate

TABLE 2A Human APP Modified Sequences SEQ SEQ SEQ Sense Sequence IDAntisense Sequence ID ID Duplex Name (5′ to 3′) NO  (5′ to 3′) NOmRNA target sequence NO AD-392699 gsasccc(Ahd)AfuUfAfAf  33asAfsagua(Ggn)gacuuaAfu  34 GUGACCCAAUUAAGUCCUACUUU  35 guccuacuuuL96Ufgggucsasc AD-392700 uscsucc(Uhd)GfaUfUfAf  36 asUfsguga(Tgn)aaauaaUfc 37 UCUCUCCUGAUUAUUUAUCACAU  38 uuuaucacauL96 Afggagasgsa AD-392703cscsuga(Ahd)CfuUfGfAf  39 asUfsggau(Tgn)aauucaAfg  40UGCCUGAACUUGAAUUAAUCCAC  41 auuaauccauL96 Ufucaggscsa AD-392704gsgsuuc(Ahd)AfaCfAfAf  42 asAfsuugc(Agn)ccuuugUfu  43UGGGUUCAAACAAAGGUGCAAUC  44 aggugcaauuL96 Ufgaaccscsa AD-392705ususuac(Uhd)CfaUfUfAf  45 csAfsaaag(Ggn)cgauaaUfg  46GAUUUACUCAUUAUCGCCUUUUG  47 ucgccuuuugL96 Afguaaasusc AD-392707asusuua(Ghd)CfuGfUfAf  48 asCfsuagu(Tgn)ugauacAfg  49GAAUUUAGCUGUAUCAAACUAGU  50 ucaaacuaguL96 Cfuaaaususc AD-392708asgsuau(Uhd)CfcUfUfUf  51 asGfsugau(Cgn)aggaaaGfg  52UAAGUAUUCCUUUCCUGAUCACU  53 ccugaucacuL96 Afauacususa AD-392709gscsuua(Uhd)GfaCfAfUf  54 gsAfsaagc(Ggn)aucaugUfc  55UUGCUUAUGACAUGAUCGCUUUC  56 gaucgcuuucL96 Afuaagcsasa AD-392710asasgau(Ghd)UfgUfCfUf  57 usAfscaaa(Tgn)ugaagaCfa  58UUAAGAUGUGUCUUCAAUUUGUA  59 ucaauuuguaL96 Cfaucuusasa AD-392711gscsaaa(Ahd)CfcAfUfUf  60 asufsagug(Agn)agcaauGfg  61CAGCAAAACCAUUGCUUCACUAC  62 gcuucacuauL96 Ufuuugcsusg AD-392712asusuua(Chd)UfcAfUfUf  63 asAfsaagg(Cgn)gauaauGfa  64UGAUUUACUCAUUAUCGCCUUUU  65 aucgccuuuuL96 Gfuaaauscsa AD-392713usascuc(Ahd)UfuAfUfCf  66 asUfscaaa(Agn)ggcgauAfa  67uuuACUCAUUAUCGCCUUUUGAC  68 gccuuuugauL96 Ufgaguasasa AD-392714usgsccu(Ghd)AfaCfUfUf  69 asGfsauua(Agn)uucaagUfu  70GAUGCCUGAACUUGAAUUAAUCC  71 guauuaaucuL96 Cfaggcasusc AD-392715csusgaa(Chd)UfuGfAfAf  72 usGfsugga(Tgn)uaauucAfa  73GCCUGAACUUGAAUUAAUCCACA  74 uuaauccacaL96 Gfuucagsgsc AD-392716ususuag(Chd)UfgUfAfUf  75 asAfscuag(Tgn)uugauaCfa  76AAUUUAGCUGUAUCAAACuAGuG  77 caaacuaguuL96 Gfcuaaasusu AD-392717gsasaua(Ghd)AfuUfCfUf  78 usAfsauca(Ggn)gagagaAfu  79AuGAAUAGAUUCUCUCCUGAUUA  80 cuccugauuaL96 Cfuauucsasu AD-392718uscscug(Ahd)UfuAfUfUf  81 asUfsaugu(Ggn)auaaauAfa  82UCUCCUGAUUAUUUAUCACAUAG  83 uaucacauauL96 Ufcaggasgsa AD-392719cscscaa(Uhd)UfaAfGfUf  84 asUfsaaag(Tgn)aggacuUfa  85GACCCAAUUAAGUCCUACUUUAC  86 ccuacuuuauL96 Afuugggsusc AD-392720csasuau(Ghd)CfuUfUfAf  87 asAfsucga(Tgn)ucuuaaAfg  88UACAUAUGCUUUAAGAAUCGAUG  89 agaaucgauuL96 Cfauaugsusa AD-392721csusucu(Chd)UfuGfCfCf  90 asGfsaaua(Cgn)uuaggcAfa  91UGCUUCUCUUGCCUAAGUAUUCC  92 uaaguauucuL96 Gfagaagscsa AD-392722csasuug(Chd)UfuAfUfGf  93 asCfsgauc(Agn)ugucauAfa  94AUCAUUGCUUAUGACAUGAUCGC  95 acaugaucguL96 Gfcaaugsasu AD-392723csusuau(Ghd)AfcAfUfGf  96 asGfsaaag(Cgn)gaucauGfu  97UGCUUAUGACAUGAUCGCUUUCU  98 aucgcuuucuL96 Cfauaagscsa AD-392724usasuga(Chd)AfuGfAfUf  99 asufsagaa(Agn)gcgaucAfu 100CuUAUGACAUGAUCGCUUUCUAC 101 cgcuuucuauL96 Gfucauasasg AD-392725usgsaca(Uhd)GfaUfCfGf 102 asUfsguag(Agn)aagcgaUfc 103UAUGACAUGAUCGCUUUCUACAC 104 ucuucuacauL96 Afugucasusa AD-392726gsasucg(Chd)UfuUfCfUf 105 asAfsuaca(Ggn)uguagaAfa 106AUGAUCGCUUUCUACACUGUAUU 107 acacuguauuL96 Gfcgaucsasu AD-392727asasaac(Uhd)AfuUfCfAf 108 asGfsacgu(Cgn)aucugaAfu 109GCAAAACUAUUCAGAUGACGUCU 110 gaugacgucuL96 Afguuuusgsc AD-392728asasacu(Ahd)UfuCfAfGf 111 asAfsgacg(Tgn)caucugAfa 112CAAAACUAUUCAGAUGACGUCUU 113 augacgucuuL96 Ufaguuususg AD-392729ascsgaa(Ahd)AfuCfCfAf 114 asCfsuugu(Agn)gguuggAfu 115CUACGAAAAUCCAACCUACAAGU 116 cacuacaaguL96 Ufuucgusasg AD-392730usgscuu(Chd)UfcUfUfGf 117 asAfsuacu(Tgn)aggcaaGfa 118GCUGCUUCUCUUGCCUAAGUAUU 119 ccuaaguauuL96 Gfaagcasgsc AD-392731usgscuu(Ahd)UfgAfCfAf 120 asAfsagcg(Agn)ucauguCfa 121AUUGCUUAUGACAUGAUCGCUUU 122 ugaucgcuuuL96 Ufaagcasasu AD-392732usgsauc(Ghd)CfuUfUfCf 123 asUfsacag(Tgn)guagaaAfg 124CAUGAUCGCUUUCUACACUGUAU 125 uacacuguauL96 Cfgaucasusg AD-392733asuscgc(Uhd)UfuCfUfAf 126 usAfsauac(Agn)guguagAfa 127UGAUCGCUUUCUACACUGUAUUA 128 cacuguauuaL96 Afgcgauscsa AD-392734uscsuuu(Ghd)AfcCfGfAf 129 asGfsuuuu(Cgn)gimucgGfu 130CAUCUUUGACCGAAACGAAAACC 131 aacgaaaacuL96 Cfaaagasusg AD-392735gsusucu(Ghd)GfgUfUfGf 132 usGfsauau(Tgn)ugucaaCfc 133AGGUUCUGGGUUGACAAAUAUCA 134 acaaauaucaL96 Cfagaacscsu AD-392736usgsggu(Uhd)GfaCfAfAf 135 asUfscuug(Agn)uauuugUfc 136UCUGGGUUGACAAAUAUCAAGAC 137 auaucaagauL96 Afacccasgsa AD-392737gsasuuu(Ahd)CfuCfAfUf 138 asAfsaggc(Ggn)auaaugAfg 139AUGAUUUACUCAUUAUCGCCUUU 140 uaucgccuuuL96 Ufaaaucsasu AD-392738uscscuu(Uhd)CfcUfGfAf 141 usGfscaua(Ggn)ugaucaGfg 142AUUCCUUUCCUGAUCACUAUGCA 143 ucacuaugcaL96 Afaaggasasu AD-392739csusuuc(Chd)UfgAfUfCf 144 asAfsugca(Tgn)agugauCfa 145UCCUUUCCUGAUCACUAUGCAUU 146 acuaugcauuL96 Gfgaaagsgsa AD-392740asusugc(Uhd)UfaUfGfAf 147 asGfscgau(Cgn)augucaUfa 148UCAUUGCUUAUGACAUGAUCGCU 149 caugaucgcuL96 Afgcaausgsa AD-392741uscsuuu(Ahd)AfcCfAfGf 150 asAfsacuu(Cgn)agacugGfu 151UUUCUUUAACCAGUCUGAAGUUU 152 ucugaaguuuL96 Ufaaagasasa AD-392742gsgsauc(Ahd)GfuUfAfCf 153 asAfsucgu(Tgn)uccguaAfc 154AAGGAUCAGUUACGGAAACGAUG 155 ggaaacgauuL96 Ufgauccsusu AD-392743csusggg(Uhd)UfgAfCfAf 156 usCfsuuga(Tgn)auuuguCfa 157UUCUGGGUUGACAAAUAUCAAGA 158 aauaucaagaL96 Afcccagsasa AD-392744asusgau(Uhd)UfaCfUfCf 159 asGfsgcga(Tgn)aaugagUfa 160UUAUGAUUUACUCAUUAUCGCCU 161 auuaucgccuL96 Afaucausasa AD-392745csusugu(Ghd)GfuUfUfGf 162 asAfsuugg(Ggn)ucacaaAfc 163UUCUUGUGGUUUGUGACCCAAUU 164 ugacccaauuL96 Cfacaagsasa AD-392746asusaug(Chd)UfuUfAfAf 165 asCfsaucg(Agn)uucuuaAfa 166ACAUAUGCUUUAAGAAUCGAuGG 167 gaaucgauguL96 Gfcauausgsu AD-392747ususugu(Chd)CfaCfGfUf 168 asCfsccaa(Agn)gauacgUfg 169UUUUUGUCCACGUAUCUUUGGGU 170 aucuuuggguL96 Gfacaaasasa AD-392748uscsauu(Ghd)UfaAfGfCf 171 asCfsguaa(Agn)agugcuUfa 172GUUCAUUGUAAGCACUUUUACGG 173 acuuuuacguL96 Cfaaugasasc AD-392749gsgscca(Ahd)CfaUfGfAf 174 asGfsuuca(Cgn)uaaucaUfg 175UUGGCCAACAUGAUUAGUGAACC 176 uuagugaacuL96 Ufuggccsasa AD-392750gsasuca(Ghd)UfuAfCfGf 177 asCfsaucg(Tgn)uuccguAfa 178AGGAUCAGUUACGGAAACGAuGc 179 gaaacgauguL96 Cfugaucscsu AD-392751usascgg(Ahd)AfaCfGfAf 180 asAfsugag(Agn)gcaucgUfu 181GUUACGGAAACGAUGCUCUCAUG 182 ugcucucauuL96 Ufccguasasc AD-392752usgsauu(Uhd)AfcUfCfAf 183 asAfsggcg(Agn)uaaugaGfu 184UAUGAUUUACUCAUUAUCGCCUU 185 uuaucgccuuL96 Afaaucasusa AD-392753gsusaga(Uhd)GfcCfUfGf 186 asAfsuuca(Agn)guucagGfc 187AAGUAGAUGCCUGAACUUGAAUU 188 aacuugaauuL96 Afucuacsusu AD-392754ususgua(Uhd)AfuUfAfUf 189 asAfsccac(Agn)agaauaAfu 190AGUUGUAUAUUAUUCUUGUGGUU 191 ucuugugguuL96 Afuacaascsu AD-392755asusugc(Uhd)GfcUfUfCf 192 asAfsauau(Agn)gcagaaGfc 193AGAUUGCUGCUUCUGCUAUAUUU 194 ugcuauauuuL96 Afgcaauscsu AD-392756usgscua(Uhd)AfuUfUfGf 195 usCfscuau(Agn)ucacaaAfu 196UCUGCUAUAUUUGUGAUAUAGGA 197 ugauauaggaL96 Afuagcasgsa AD-392757ascsaca(Uhd)UfaGfGfCf 198 asAfsgucu(Cgn)aaugccUfa 199GCACACAUUAGGCAUUGAGACUU 200 auugagacuuL96 Afugugusgsc AD-392758asasgaa(Uhd)CfcCfUfGf 201 usUfsacaa(Tgn)gaacagGfg 202AAAAGAAUCCCUGUUCAUUGUAA 203 uucauuguaaL96 Afuucuususu AD-392759csasuug(Uhd)AfaGfCfAf 204 asCfscgua(Agn)aagugcUfu 205UUCAUUGUAAGCACUUUUACGGG 206 cuuuuacgguL96 Afcaaugsasa AD-392760ususgcu(Uhd)AfuGfAfCf 207 asAfsgcga(Tgn)caugucAfu 208CAUUGCUUAUGACAUGAUCGCUU 209 augaucgcuuL96 Afagcaasusg AD-392761csasagg(Ahd)UfcAfGfUf 210 asGfsuuuc(Cgn)guaacuGfa 211ACCAAGGAUCAGUUACGGAAACG 212 uacggaaacuL96 Ufccuugsgsu AD-392762asgsguu(Chd)UfgGfGfUf 213 asUfsauuu(Ggn)ucaaccCfa 214CCAGGUUCUGGGUUGACAAAUAU 215 ugacaaauauL96 Gfaaccusgsg AD-392763asasgau(Ghd)UfgGfGfUf 216 asUfsuugu(Tgn)ugaaccCfa 217AGAAGAUGUGGGUUCAAACAAAG 218 ucaaacaaauL96 Cfaucuuscsu AD-392764csusgaa(Ghd)AfaGfAfAf 219 usGfsugua(Cgn)uguuucUfu 220UGCUGAAGAAGAAACAGUACACA 221 acaguacacaL96 Cfuucagscsa AD-392765asasguu(Ghd)GfaCfAfGf 222 asAfsuggu(Tgn)uugcugUfc 223UGAAGUUGGACAGCAAAACCAUU 224 caaaaccauuL96 Cfaacuuscsa AD-392766asuscgg(Uhd)GfuCfCfAf 225 asUfsucua(Tgn)aaauggAfc 226CCAUCGGUGUCCAUUUAUAGAAU 227 uuuauagaauL96 Afccgausgsg AD-392767uscsggu(Ghd)UfcCfAfUf 228 usAfsuucu(Agn)uaaaugGfa 229CAUCGGUGUCCAUUUAUAGAAUA 230 uuauagaauaL96 Cfaccgasusg AD-392768gscsugu(Ahd)AfcAfCfAf 231 asGfscauc(Tgn)acuuguGfu 232GUGCUGUAACACAAGUAGAUGCC 233 aguagaugcuL96 Ufacagcsasc AD-392769asasgua(Ghd)AfuGfCfCf 234 usUfscaag(Tgn)ucaggcAfu 235ACAAGUAGAUGCCUGAACUUGAA 236 ugaacuugaaL96 Cfuacuusgsu AD-392770ususgug(Ghd)UfuUfGfUf 237 usAfsauug(Ggn)gucacaAfa 238UCUUGUGGUUUGUGACCCAAUUA 239 gacccaauuaL96 Cfcacaasgsa AD-392771gsusuug(Uhd)GfaCfCfCf 240 asGfsacuu(Agn)auugggUfc 241UGGUUUGUGACCCAAUUAAGUCC 242 aauuaagucuL96 Afcaaacscsa AD-392772gsusgac(Chd)CfaAfUfUf 243 asGfsuagg(Agn)cuuaauUfg 244UUGUGACCCAAUUAAGUCCUACU 245 aaguccuacuL96 Gfgucacsasa AD-392773usasugc(Uhd)UfuAfAfGf 246 asCfscauc(Ggn)auucuuAfa 247CAUAUGCUUUAAGAAUCGAUGGG 248 aaucgaugguL96 Afgcauasusg AD-392774ususugu(Ghd)AfuAfUfAf 249 usCfsuuaa(Tgn)uccuauAfu 250UAUUUGUGAUAUAGGAAUUAAGA 251 ggaauuaagaL96 Cfacaaasusa AD-392775asasaga(Ahd)UfcCfCfUf 252 usAfscaau(Ggn)aacaggGfa 253GAAAAGAAUCCCUGUUCAUUGUA 254 guucauuguaL96 Ufucuuususc AD-392776usgsauu(Ghd)UfaCfAfGf 255 asGfscaau(Ggn)auucugUfa 256GAUGAUUGUACAGAAUCAUUGCU 257 aaucauugcuL96 Cfaaucasusc AD-392777usgsccu(Ghd)GfaCfAfAf 258 asAfsagaa(Ggn)gguuugUfc 259CAUGCCUGGACAAACCCUUCUUU 260 acccuucuuuL96 Cfaggcasusg AD-392778gsasgca(Ahd)AfaCfUfAf 261 asUfscauc(Tgn)gaauagUfu 262AAGAGCAAAACUAUUCAGAUGAC 263 uucagaugauL96 Ufugcucsusu AD-392779asgsuga(Ahd)CfcAfAfGf 264 asUfsaacu(Ggn)auccuuGfg 265UUAGUGAACCAAGGAUCAGUUAC 266 gaucaguuauL96 Ufucacusasa AD-392780usgsaac(Chd)AfaGfGfAf 267 asCfsguaa(Cgn)ugauccUfu 268AGUGAACCAAGGAUCAGUUACGG 269 ucaguuacguL96 Gfguucascsu AD-392781csasguu(Ahd)CfgGfAfAf 270 asGfsagca(Tgn)cguuucCfg 271AUCAGUUACGGAAACGAUGCUCU 272 acgaugcucuL96 Ufaacugsasu AD-392782asgsaag(Ahd)UfgUfGfGf 273 usUfsguuu(Ggn)aacccaCfa 274GCAGAAGAUGUGGGUUCAAACAA 275 guucaaacaaL96 Ufcuucusgsc AD-392783cscsucu(Ghd)AfaGfUfUf 276 usUfsugcu(Ggn)uccaacUfu 277AGCCUCUGAAGUUGGACAGCAAA 278 ggacagcaaaL96 Cfagaggscsu AD-392784ususaug(Ahd)UfuUfAfCf 279 ascfsgaua(Agn)ugaguaAfa 280UUUUAUGAUUUACUCAUUAUCGC 281 ucauuaucguL96 Ufcauaasasa AD-392785ascsagc(Uhd)GfuGfCfUf 282 asUfsugug(Tgn)uacagcAfc 283UGACAGCUGUGCUGUAACACAAG 284 guaacacaauL96 Afgcuguscsa AD-392786usgsuga(Chd)CfcAfAfUf 285 asUfsagga(Cgn)uuaauuGfg 286UUUGUGACCCAAUUAAGUCCUAC 287 uaaguccuauL96 Gfucacasasa AD-392787usascau(Ahd)UfgCfUfUf 288 usCfsgauu(Cgn)uuaaagCfa 289UUUACAUAUGCUUUAAGAAUCGA 290 uaagaaucgaL96 Ufauguasasa AD-392788gsusaaa(Uhd)AfaAfUfAf 291 usCfscaag(Agn)auguauUfu 292AUGUAAAUAAAUACAUUCUUGGA 293 cauucuuggaL96 Afuuuacsasu AD-392789uscsagu(Uhd)AfcGfGfAf 294 asAfsgcau(Cgn)guuuccGfu 295GAUCAGUUAGGGAAACGAUGCUC 296 aacgaugcuuL96 Afacugasusc AD-392790csusucc(Chd)GfuGfAfAf 297 asAfsacuc(Tgn)ccauucAfc 298UCCUUCCCGUGAAUGGAGAGUUC 299 uggagaguuuL96 Gfggaagsgsa AD-392791asgsuug(Ghd)AfcAfGfCf 300 asAfsaugg(Tgn)uuugcuGfu 301GAAGUUGGACAGCAAAACCAUUG 302 aaaaccauuuL96 Cfcaacususc AD-392792cscscau(Chd)GfgUfGfUf 303 asUfsauaa(Agn)uggacaCfc 304UACCCAUCGGUGUCCAUUUAUAG 305 ccauuuauauL96 Gfaugggsusa AD-392793usgscac(Ahd)CfaUfUfAf 306 usCfsucaa(Tgn)gccuaaUfg 307UGUGCACACAUUAGGCAUUGAGA 308 ggcauugagaL96 Ufgugcascsa AD-392794cscsaac(Ahd)UfgAfUfUf 309 usufsgguu(Cgn)acuaauCfa 310GGCCAACAUGAUUAGUGAACCAA 311 agugaaccaaL96 Ufguuggscsc AD-392795asusgau(Uhd)AfgUfGfAf 312 asAfsuccu(Tgn)gguucaCfu 313ACAUGAUUAGUGAACCAAGGAUC 314 accaaggauuL96 Afaucausgsu AD-392796ususagu(Ghd)AfaCfCfAf 315 asAfscuga(Tgn)ccuuggUfu 316GAUUAGUGAACCAAGGAUCAGUU 317 aggaucaguuL96 Cfacuaasusc AD-392797asascca(Ahd)GfgAfUfCf 318 usUfsccgu(Agn)acugauCfc 319UGAACCAAGGAUCAGUUACGGAA 320 aguuacggaaL96 Ufugguuscsa AD-392798gsusuac(Ghd)GfaAfAfCf 321 usGfsagag(Cgn)aucguuUfc 322CAGUUACGGAAACGAUGCUCUCA 323 gaugcucucaL96 Cfguaacsusg AD-392799gsasugc(Ahd)GfaAfUfUf 324 asUfscaug(Tgn)cggaauUfc 325uGGAUGCAGAAUUCCGACAUGAC 326 ccgacaugauL96 Ufgcaucscsa AD-392800ususgga(Chd)AfgCfAfAf 327 asGfscaau(Ggn)guuuugCfu 328AGUUGGACAGCAAAACCAUUGCU 329 aaccauugcuL96 Gfuccaascsu AD-392801asasacc(Ahd)UfuGfCfUf 330 usGfsggua(Ggn)ugaagcAfa 331CAAAACCAUUGCUUCACUACCCA 332 ucacuacccaL96 Ufgguuususg AD-392802cscsauc(Ghd)GfuGfUfCf 333 usCfsuaua(Agn)auggacAfc 334ACCCAUCGGUGUCCAUUUAUAGA 335 cauuuauagaL96 Cfgauggsgsu AD-392803ususauc(Ghd)CfcUfUfUf 336 asCfsagcu(Ggn)ucaaaaGfg 337CAUUAUCGCCUUUUGACAGCUGU 338 ugacagcuguL96 Cfgauaasusg AD-392804asuscgc(Chd)UfuUfUfGf 339 asCfsacag(Cgn)ugucaaAfa 340UUAUCGCCUUUUGACAGCUGUGC 341 acagcuguguL96 Gfgcgausasa AD-392805ascsaca(Ahd)GfuAfGfAf 342 asGfsuuca(Ggn)gcaucuAfc 343UAACACAAGUAGAUGCCUGAACU 344 ugccugaacuL96 Ufugugususa AD-392806usgsugg(Uhd)UfuGfUfGf 345 usUfsaauu(Ggn)ggucacAfa 346CUUGUGGUUUGUGACCCAAUUAA 347 acccaauuaaL96 Afccacasasg AD-392807gsgsgau(Ghd)CfuUfCfAf 348 asAfscguu(Cgn)acaugaAfg 349GGGGGAUGCUUCAUGUGAACGUG 350 ugugaacguuL96 Cfaucccscsc AD-392808usgsugc(Ahd)CfaCfAfUf 351 usCfsaaug(Cgn)cuaaugUfg 352UAUGUGCACACAUUAGGCAUUGA 353 uaggcauugaL96 Ufgcacasusa AD-392809asasaug(Ghd)AfaGfUfGf 354 asufsuaua(rgn)ugccacUfu 355GAAAAUGGAAGUGGCAAUAUAAG 356 gcaauauaauL96 Cfcauuususc AD-392810asusgga(Ahd)GfuGfGfCf 357 asCfscuua(Tgn)auugccAfc 358AAAUGGAAGUGGCAAUAUAAGGG 359 aauauaagguL96 Ufuccaususu AD-392811usgsccc(Ghd)AfgAfUfCf 360 asGfsuuua(Agn)caggauCfu 361CUUGCCCGAGAUCCUGUUAAACU 362 cuguuaaacuL96 Cfgggcasasg AD-392812asusuag(Uhd)GfaAfCfCf 363 asCfsugau(Cgn)cuugguUfc 364UGAUUAGUGAACCAAGGAUCAGU 365 aaggaucaguL96 Afcuaauscsa AD-392813gsasacc(Ahd)AfgGfAfUf 366 usCfscgua(Agn)cugaucCfu 367GUGAACCAAGGAUCAGUUACGGA 368 caguuacggaL96 Ufgguucsasc AD-392814asasgga(Uhd)CfaGfUfUf 369 usCfsguuu(Cgn)cguaacUfg 370CCAAGGAUCAGUUACGGAAACGA 371 acggaaacgaL96 Afuccuusgsg AD-392815csasaca(Chd)AfgAfAfAf 372 usCfsaacu(Tgn)cguuuuCfu 373GCCAACACAGAAAACGAAGUUGA 374 acgaaguugaL96 Gfuguugsgsc AD-392816usgsggu(Uhd)CfaAfAfCf 375 usUfsgcac(Cgn)uuuguuUfg 376UGUGGGUUCAAACAAAGGUGCAA 377 aaaggugcaaL96 Afacccascsa AD-392817csasgug(Ahd)UfcGfUfCf 378 asCfsaagg(Tgn)gaugacGfa 379GACAGUGAUCGUCAUCACCUUGG 380 aucaccuuguL96 Ufcacugsusc AD-392818ascscca(Uhd)CfgGfUfGf 381 usAfsuaaa(Tgn)ggacacCfg 382CUACCCAUCGGUGUCCAUUUAUA 383 uccauuuauaL96 Afugggusasg AD-392819uscsuug(Uhd)GfgUfUfUf 384 asUfsuggg(Tgn)cacaaaCfc 385AUUCUUGUGGUUUGUGACCCAAU 386 gugacccaauL96 Afcaagasasu AD-392820ususugu(Ghd)AfcCfCfAf 387 asGfsgacu(Tgn)aauuggGfu 388GGUUUGUGACCCAAUUAAGUCCU 389 auuaaguccuL96 Cfacaaascsc AD-392821ususgug(Ahd)CfcCfAfAf 390 usAfsggac(Tgn)uaauugGfg 391GUUUGUGACCCAAUUAAGUCCUA 392 uuaaguccuaL96 Ufcacaasasc AD-392822ususcag(Ahd)UfgAfCfGf 393 usUfsggcc(Agn)agacguCfa 394UAUUCAGAUGACGUCUUGGCCAA 395 ucuuggccaaL96 Ufcugaasusa AD-392823asuscag(Uhd)UfaCfGfGf 396 asGfscauc(Ggn)uuuccgUfa 397GGAUCAGUUACGGAAACGAUGCU 398 aaacgaugcuL96 Afcugauscsc AD-392824usgsgau(Ghd)CfaGfAfAf 399 asAfsuguc(Ggn)gaauucUfg 400GAUGGAUGCAGAAUUCCGACAUG 401 uuccgacauuL96 Cfauccasusc AD-392825gsuscca(Ahd)GfaUfGfCf 402 asCfsgtmc(Tgn)gcugcaUfc 403CUGUCCAAGAUGCAGCAGAACGG 404 agcagaacguL96 Ufuggacsasg AD-392826usasccc(Ahd)UfcGfGfUf 405 asUfsaaau(Ggn)gacaccGfa 406ACUACCCAUCGGUGUCCAUUUAU 407 guccauuuauL96 Ufggguasgsu AD-392827ususuug(Ahd)CfaGfCfUf 408 asUfsuaca(Ggn)cacagcUfg 409CCUUUUGACAGCUGUGCUGUAAC 410 gugcuguaauL96 Ufcaaaasgsg AD-392828ususgac(Ahd)GfcUfGfUf 411 asUfsgtua(Cgn)agcacaGfc 412UUUUGACAGCUGUGCUGUAACAC 413 gcuguaacauL96 Ufgucaasasa AD-392829asgscug(Uhd)GfcUfGfUf 414 usAfscuug(Tgn)guuacaGfc 415ACAGCUGUGCUGUAACACAAGUA 416 aacacaaguaL96 Afcagcusgsu AD-392830gsusuuu(Ahd)UfgUfGfCf 417 asCfsuaau(Ggn)ugugcaCfa 418CUGUUUUAUGUGCACACAUUAGG 419 acacauuaguL96 Ufaaaacsasg AD-392831ususcaa(Uhd)UfaCfCfAf 420 asGfsagaa(Tgn)ucuuggUfa 421UCUUCAAUUACCAAGAAUUCUCC 422 agaauucucuL96 Afuugaasgsa AD-392832csascac(Ahd)UfcAfGfUf 423 asGfsaaua(Cgn)auuacuGfa 424UCCACACAUCAGUAAUGUAUUCU 425 aauguauucuL96 Ufgugugsgsa AD-392833usgsguc(Uhd)CfuAfUfAf 426 asAfsuaau(Ggn)uaguauAfg 427UUUGGUCUCUAUACUACAUUAUU 428 cuacauuauuL96 Afgaccasasa AD-392834ascsccg(Uhd)UfuUfAfUf 429 usGfsagua(Agn)aucauaAfa 430AAACCCGUUUUAUGAUUUACUCA 431 gauuuacucaL96 Afcgggususu AD-392835usascga(Ahd)AfaUfCfCf 432 asUfsugua(Ggn)guuggaUfu 433GCUACGAAAAUCCAACCUACAAG 434 aaccuacaauL96 Ufucguasgsc AD-392836uscscac(Ahd)CfaUfCfAf 435 asAfsuaca(Tgn)uacugaUfg 436AAUCCACACAUCAGUAAUGUAUU 437 guaauguauuL96 Ufguggasusu AD-392837csusggu(Chd)UfuCfAfAf 438 usUfscuug(Ggn)uaauugAfa 439UGCUGGUCUUCAAUUACCAAGAA 440 uuaccaagaaL96 Gfaccagscsa AD-392838gscscau(Chd)UfuUfGfAf 441 usUfscguu(Tgn)cggucaAfa 442AUGCCAUCUUUGACCGAAACGAA 443 ccgaaacgaaL96 Gfauggcsasu AD-392839cscsauc(Uhd)UfuGfAfCf 444 usUfsucgu(Tgn)ucggucAfa 445UGCCAUCUUUGACCGAAACGAAA 446 cgaaacgaaaL96 Afgauggscsa AD-392840csusacg(Ahd)AfaAfUfCf 447 usUfsguag(Ggn)uuggauUfu 448GGCUACGAAAAUCCAACCUACAA 449 caaccuacaaL96 Ufcguagscsc AD-392841asuscca(Chd)AfcAfUfCf 450 asUfsacau(Tgn)acugauGfu 451uAAUCCACACAUCAGUAAUGUAU 452 aguaauguauL96 Gfuggaususa AD-392842csasugc(Chd)AfuCfUfUf 453 asUfsuucg(Ggn)ucaaagAfu 454CUCAUGCCAUCUUUGACCGAAAC 455 ugaccgaaauL96 Gfgcaugsasg AD-392843gsgscua(Chd)GfaAfAfAf 456 asUfsaggu(Tgn)ggauuuUfc 457ACGGCUACGAAAAUCCAACCUAC 458 uccaaccuauL96 Gfuagccsgsu AD-392844uscsaug(Chd)CfaUfCfUf 459 usUfsucgg(Tgn)caaagaUfg 460UCUCAUGCCAUCUUUGACCGAAA 461 uugaccgaaaL96 Gfcaugasgsa AD-392845csasgua(Chd)AfcAfUfCf 462 asUfsgaug(Agn)auggauGfu 463AACAGUACACAUCCAUUCAUCAU 464 cauucaucauL96 Gfuacugsusu AD-392846asascgg(Chd)UfaCfGfAf 465 asGfsuugg(Agn)uuuucgUfa 466AGAACGGCUACGAAAAUCCAACC 467 aaauccaacuL96 Gfccguuscsu AD-392847gsasagu(Uhd)UfcAfUfUf 468 usUfsguau(Cgn)auaaauGfa 469CUGAAGUUUCAUUUAUGAUACAA 470 uaugauacaaL96 Afacuucsasg AD-392848asusgcc(Ahd)UfcUfUfUf 471 asGfsuuuc(Ggn)gucaaaGfa 472UCAUGCCAUCUUUGACCGAAACG 473 gaccgaaacuL96 Ufggcausgsa AD-392849gsasacg(Ghd)CfuAfCfGf 474 asUfsugga(Tgn)uuucguAfg 475CAGAACGGCUACGAAAAUCCAAC 476 aaaauccaauL96 Cfcguucsusg AD-392850uscsuuc(Ghd)UfgCfCfUf 477 asAfscaua(Agn)aacaggCfa 478UUUCUUCGUGCCUGUUUUAUGUG 479 guuuuauguuL96 Cfgaagasasa AD-392851ususgcc(Chd)GfaGfAfUf 480 asUfsuuaa(Cgn)aggaucUfc 481UCUUGCCCGAGAUCCUGUUAAAC 482 ccuguuaaauL96 Gfggcaasgsa AD-392852csusucg(Uhd)GfcCfUfGf 483 asCfsacau(Agn)aaacagGfc 484UUCUUCGUGCCUGUUUUAUGUGC 485 uuuuauguguL96 Afcgaagsasa AD-392853gscsgcc(Ahd)UfgUfCfCf 486 asUfsaaac(Tgn)uugggaCfa 487CAGCGCCAUGUCCCAAAGUUUAC 488 caaaguuuauL96 Ufggcgcsusg AD-392854gsuscau(Ahd)GfcGfAfCf 489 asAfscgau(Cgn)acugucGfc 490UUGUCAUAGCGACAGUGAUCGUC 491 agugaucguuL96 Ufaugacsasa AD-392855gscsuac(Ghd)AfaAfAfUf 492 usGfsuagg(Tgn)uggautfau 493CGGCUACGAAAAUCCAACCUACA 494 ccaaccuacaL96 Cfguagcscsg AD-392856asusagc(Ghd)AfcAfGfUf 495 asAfsugac(Ggn)aucacuGfu 496UCAUAGCGACAGUGAUCGUCAUC 497 gaucgucauuL96 Cfgcuausgsa AD-392857csusugc(Chd)CfgAfGfAf 498 usUfsuaac(Agn)ggaucuCfg 499CUCUUGCCCGAGAUCCUGUUAAA 500 uccuguuaaaL96 Gfgcaagsasg AD-392858csuscau(Ghd)CfcAfUfCf 501 usUfscggu(Cgn)aaagauGfg 502CUCUCAUGCCAUCUUUGACCGAA 503 uuugaccgaaL96 Cfaugagsasg AD-392859ascsggc(Uhd)AfcGfAfAf 504 asGfsguug(Ggn)auuuucGfu 505GAACGGCUACGAAAAUCCAACCu 506 aauccaaccuL96 Afgccgususc AD-392860csasuca(Ahd)AfaAfUfUf 507 asAfsagaa(Cgn)accaauUfu 508AUCAUCAAAAAUUGGUGUUCUUU 509 gguguucuuuL96 Ufugaugsasu AD-392861asuscca(Ahd)CfcUfAfCf 510 csAfsaaga(Agu)cuuguaGfg 511AAAUCCAACCUACAAGUUCUUUG 512 aaguucuuugL96 Ufuggaususu AD-392862csgscuu(Uhd)CfuAfCfAf 513 usGfsuaau(Agn)caguguAfg 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AD-392871ususcua(Chd)AfcUfGfUf 540 usUfsuaug(Tgn)aauacaGfu 541CUUUCUACACUGUAUUACAUAAA 542 auuacauaaaL96 Gfuagaasasg AD-392872uscsuac(Ahd)CfuGfUfAf 543 asUfsuuau(Ggn)uaauacAfg 544UUUCUACACUGUAUUACAUAAAU 545 uuacauaaauL96 Ufguagasasa AD-392873csusuuu(Ahd)AfgAfUfGf 546 asUfsugaa(Ggn)acacauCfu 547UUCUUUUAAGAUGUGUCUUCAAU 548 ugucuucaauL96 Ufaaaagsasa AD-392874asusgug(Uhd)CfuUfCfAf 549 usUfsauac(Agn)aauugaAfg 550AGAUGUGUCUUCAAUUUGUAUAA 551 auuuguauaaL96 Afcacauscsu AD-392875asuscaa(Ahd)AfaUfUfGf 552 csAfsaaga(Agn)caccaaUfu 553UCAUCAAAAAUUGGUGUUCUUUG 554 guguucuuugL96 Ufuugausgsa AD-392876asasauc(Chd)AfaCfCfUf 555 asAfsgaac(Tgn)uguaggUfu 556GAAAAUCCAACCUACAAGUUCUU 557 acaaguucuuL96 Gfgauuususc AD-392877gsusacu(Ghd)UfaAfAfGf 558 asAfsgcua(Agn)auucuuUfa 559GUGUACUGUAAAGAAUUUAGCUG 560 aauuuagcuuL96 Cfaguacsasc AD-392878csusccu(Ghd)AfuUfAfUf 561 usAfsugug(Agn)uaaauaAfu 562CUCUCCUGAUUAUUUAUCACAUA 563 uuaucacauaL96 Cfaggagsasg AD-392879gscscag(Uhd)UfgUfAfUf 564 asAfsagaa(Tgn)aauauaCfa 565UAGCCAGUUGUAUAUUAUUCUUG 566 auuauucuuuL96 Afcuggcsusa AD-392880asasuua(Ahd)GfuCfCfUf 567 usAfsugua(Agn)aguaggAfc 568CCAAUUAAGUCCUACUUUACAUA 569 acuuuacauaL96 Ufuaauusgsg AD-392881csusugc(Chd)UfaAfGfUf 570 asGfsaaag(Ggn)aauacuUfa 571CUCUUGCCUAAGUAUUCCUUUCC 572 auuccuuucuL96 Gfgcaagsasg AD-392882asusucc(Uhd)UfuCfCfUf 573 asAfsuagu(Ggn)aucaggAfa 574GUAUUCCUUUCCUGAUCACUAUG 575 gaucacuauuL96 Afggaausasc AD-392883ascsuau(Ghd)CfaUfUfUf 576 usUfsuaac(Tgn)uuaaaaUfg 577UCACUAUGCAUUUUAAAGUUAAA 578 uaaaguuaaaL96 Cfauagusgsa AD-392884usgsuuc(Ahd)UfuGfUfAf 579 usAfsaaag(Tgn)gcuuacAfa 580CCUGUUCAUUGUAAGCACUUUUA 581 agcacuuuuaL96 Ufgaacasgsg AD-392885asasuua(Chd)CfaAfGfAf 582 usUfsugga(Ggn)aauucuUfg 583UCAAUUACCAAGAAUUCUCCAAA 584 auucuccaaaL96 Gfuaauusgsa AD-392886ususacc(Ahd)AfgAfAfUf 585 asUfsuuug(Ggn)agaauuCfu 586AAUUACCAAGAAUUCUCCAAAAC 587 ucuccaaaauL96 Ufgguaasusu AD-392887uscsauu(Ghd)CfuUfAfUf 588 asGfsauca(Tgn)gucauaAfg 589AAUCAUUGCUUAUGACAUGAUCG 590 gacaugaucuL96 Cfaaugasusu AD-392889ususuua(Ahd)GfaUfGfUf 591 asAfsuuga(Agn)gacacaUfc 592UCUUUUAAGAUGUGUCUUCAAUU 593 gucuucaauuL96 Ufuaaaasgsa AD-392890asusccu(Ghd)UfuAfAfAf 594 usUfsguag(Ggn)aaguuuAfa 595AGAUCCUGUUAAACUUCCUACAA 596 cuuccuacaaL96 Cfaggauscsu AD-392891ascsuau(Uhd)CfaGfAfUf 597 asCfsaaga(Cgn)gucaucUfg 598AAACUAUUCAGAUGACGUCUUGG 599 gacgucuuguL96 Afauagususu AD-392892gsusuca(Uhd)CfaUfCfAf 600 asAfsccaa(Tgn)uuuugaUfg 601AAGUUCAUCAUCAAAAAUUGGUG 602 aaaauugguuL96 Afugaacsusu AD-392893usasucu(Chd)UfcUfUfUf 603 asCfscaaa(Agn)uguaaaGfa 604UCUAUCUCUCUUUACAUUUUGGU 605 acauuuugguL96 Gfagauasgsa AD-392894asuscuc(Uhd)CfuUfUfAf 606 asAfsccaa(Agn)auguaaAfg 607CUAUCUCUCUUUACAUUUUGGUC 608 cauuuugguuL96 Afgagausasg AD-392895usgsugu(Ahd)CfuGfUfAf 609 asUfsaaau(Tgn)cuuuacAfg 610UUUGUGUACUGUAAAGAAUUUAG 611 aagaauuuauL96 Ufacacasasa AD-392896csusacu(Uhd)UfaCfAfUf 612 asUfsuaaa(Ggn)cauaugUfa 613UCCUACUUUACAUAUGCUUUAAG 614 augcuuuuauL96 Afaguagsgsa AD-392897usgsccu(Ahd)AfgUfAfUu 615 asAfsggaa(Agn)ggaauaCfu 616CUUGCCUAAGUAUUCCUUUCCUG 617 uccuuuccuuL96 Ufaggcasasg AD-392898asasgua(Uhd)UfcCfUfUf 618 asUfsgauc(Agn)ggaaagGfa 619CUAAGUAUUCCUUUCCUGAUCAC 620 uccugaucauL96 Afuacuusasg AD-392899gsusauu(Chd)CfuUfUfCf 621 usAfsguga(Tgn)caggaaAfg 622AAGUAUUCCUUUCCUGAUCACUA 623 cugaucacuaL96 Gfaauacsusu AD-392900ususccu(Ghd)AfuCfAfCf 624 asAfsaaug(Cgn)auagugAfu 625CUUUCCUGAUCACUAUGCAUUUU 626 uauacauuuuL96 Cfaggaasasa AD-392901csusgau(Chd)AfcUfAfUf 627 usUfsuaaa(Agn)ugcauaGfu 628UCCUGAUCACUAUGCAUUUUAAA 629 gcauuuuaaaL96 Gfaucagsgsa AD-392902csascgu(Ahd)UfcUfUfUf 630 usCfsaaag(Agn)cccaaaGfa 631UCCACGUAUCUUUGGGUCUUUGA 632 gggucuuugaL96 Ufacgugsgsa AD-392903usgsggu(Chd)UfuUfGfAf 633 asUfsuuuc(Tgn)uuaucaAfa 634UUUGGGUCUUUGAUAAAGAAAAG 635 uaaagaaaauL96 Gfacccasasa AD-392904uscsaau(Uhd)AfcCfAfAf 636 usGfsgaga(Agn)uucuugGfu 637CUUCAAUUACCAAGAAUUCUCCA 638 gaauucuccaL96 Afauugasasg AD-392906uscsgcu(Uhd)UfcUfAfCf 639 asUfsaaua(Cgn)aguguaGfa 640GAUCGCUUUCUACACUGUAUUAC 641 acuguauuauL96 Afagcgasusc AD-392907asusuuu(Chd)UfuUfAfAf 642 usUfscaga(Cgn)ugguuaAfa 643CAAUUUUCUUUAACCAGUCUGAA 644 ccagucugaaL96 Gfaaaaususg AD-392908csusuua(Ahd)CfcAfGfUf 645 gsAfsaacu(Tgn)cagacuGfg 646UUCUUUAACCAGUCUGAAGUUUC 647 cugaaguuucL96 Ufuaaagsasa AD-392909usasaga(Uhd)GfuGfUfCf 648 asCfsaaau(Tgn)gaagacAfc 649UUUAAGAUGUGUCUUCAAUUUGU 650 uucaauuuguL96 Afucuuasasa AD-392910gsasucc(Uhd)GfuUfAfAf 651 usGfsuagg(Agn)aguuuaAfc 652GAGAUCCUGUUAAACUUCCUACA 653 acuuccuacaL96 Afggaucsusc AD-392911csusgcu(Uhd)CfaGfAfAf 654 asUfsuuug(Cgn)ucuuucUfg 655AGCUGCUUCAGAAAGAGCAAAAC 656 agagcaaaauL96 Afagcagscsu AD-392912csasgaa(Ahd)GfaGfCfAf 657 usGfsaaua(Ggn)uuuugcUfc 658uUCAGAAAGAGCAAAACUAUUCA 659 aaacuauucaL96 Ufuucugsasa AD-392913usasuga(Ahd)GfuUfCfAf 660 usUfsuuug(Agn)ugaugaAfc 661GAUAUGAAGUUCAUCAUCAAAAA 662 ucaucaaaaaL96 Ufucauasusc AD-392914csasuca(Uhd)CfaAfAfAf 663 asAfsacac(Cgn)aauuuuUfg 664UUCAUCAUCAAAAAUUGGUGUUC 665 auugguguuuL96 Afugaugsasa AD-392915uscsaaa(Ahd)AfuUfGfGf 666 asCfsaaag(Agn)acaccaAfu 667CAUCAAAAAUUGGUGUUCUUUGC 668 uguucuuuguL96 Ufuuugasusg AD-392916asasaau(Chd)CfaAfCfCf 669 asGfsaacu(Tgn)guaggaUfg 670CGAAAAUCCAACCUACAAGUUCU 671 uacaaguucuL96 Gfauuuuscsg AD-392917cscsaac(Chd)UfaCfAfAf 672 asUfscaaa(Ggn)aacuugUfa 673AUCCAACCUACAAGUUCUUUGAG 674 guucuuugauL96 Gfguuggsasu AD-392918ascsuca(Uhd)UfaUfCfGf 675 usGfsucaa(Agn)aggcgaUfa 676UUACUCAUUAUCGCCUUUUGACA 677 ccuuuugacaL96 Afugagusasa AD-392919csuscau(Uhd)AfuCfGfCf 678 asUfsguca(Agn)aaggcgAfu 679UACUCAUUAUCGCCUUUUGACAG 680 cuuuugacauL96 Afaugagsusa AD-392920usgsugc(Uhd)GfuAfAfCf 681 asUfscuac(Tgn)uguguuAfc 682GCUGUGCUGUAACACAAGUAGAU 683 acaaguagauL96 Afgcacasgsc AD-392921gsusgcu(Ghd)UfaAfCfAf 684 asAfsucua(Cgn)uuguguUfa 685CUGUGCUGUAACACAAGUAGAUG 686 caaguagauuL96 Cfagcacsasg AD-392922uscsuuu(Ahd)CfaUfUfUf 687 asUfsagag(Agn)ccaaaaUfg 688UCUCUUUACAUUUUGGUCUCUAU 689 uggucucuauL96 Ufaaagasgsa AD-392923asusggg(Uhd)UfuUfGfUf 690 usUfsuaca(Ggn)uacacaAfa 691UAAUGGGUUUUGUGUACUGUAAA 692 guacuguaaaL96 Afcccaususa AD-392924ususgug(Uhd)AfcUfGfUf 693 usAfsaauu(Cgn)uuuacaGfu 694UUUUGUGUACUGUAAAGAAUUUA 695 aaagaauuuaL96 Afcacaasasa AD-392925gscsugu(Ahd)UfcAfAfAf 696 asAfsugca(Cgn)uaguuuGfa 697UAGCUGUAUCAAACUAGUGCAUG 698 cuagugcauuL96 Ufacagcsusa AD-392926csusagu(Ghd)CfaUfGfAf 699 asAfsgaau(Cgn)uauucaUfg 700AACUAGUGCAUGAAUAGAUUCUC 701 auagauucuuL96 Cfacuagsusu AD-392927usasgug(Chd)AfuGfAfAf 702 asGfsagaa(Tgn)cuauucAfu 703ACUAGUGCAUGAAUAGAUUCUCU 704 uagauucucuL96 Gfcacuasgsu AD-392928csuscuc(Chd)UfgAfUfUf 705 usGfsugau(Agn)aauaauCfa 706UUCUCUCCUGAUUAUUUAUCACA 707 auuuaucacaL96 Gfgagagsasa AD-392929cscsuga(Uhd)UfaUfUfUf 708 asCfsuaug(Tgn)gauaaaUfa 709CUCCUGAUUAUUUAUCACAUAGC 710 aucacauaguL96 Afucaggsasg AD-392930usasagu(Chd)CfuAfCfUf 711 asCfsauau(Ggn)uaaaguAfg 712AUUAAGUCCUACUUUACAUAUGC 713 uuacauauguL96 Gfacuuasasu AD-392931asgsucc(Uhd)AfcUfUfUf 714 asAfsgcau(Agn)uguaaaGfu 715UAAGUCCUACUUUACAUAUGCUU 716 acauaugcuuL96 Afggacususa AD-392932gsusccu(Ahd)CfuUTUfAf 717 asAfsagca(Tgn)auguaaAfg 718AAGUCCUACUUUACAUAUGCUUU 719 cauaugcuuuL96 Ufaggacsusu AD-392933ususcuc(Uhd)UfgCfCfUf 720 asGfsgaau(Agn)cuuaggCfa 721GCUUCUCUUGCCUAAGUAUUCCU 722 aaguauuccuL96 Afgagaasgsc AD-392934csuscuu(Ghd)CfcUfAfAf 723 asAfsagga(Agn)uacuuaGfg 724UUCUCUUGCCUAAGUAUUCCUUU 725 guauuccuuuL96 Cfaagagsasa AD-392935usasuuc(Chd)UfuUfCfCf 726 asUfsagug(Agn)ucaggaAfa 727AGUAUUCCUUUCCUGAUCACUAU 728 ugaucacuauL96 Gfgaauascsu AD-392936ususucc(Uhd)GfaUfCfAf 729 asAfsaugc(Agn)uagugaUfc 730CCUUUCCUGAUCACUAUGCAUUU 731 cuaugcauuuL96 Afggaaasgsg AD-392937csascua(Uhd)GfcAfUfUf 732 usUfsaacu(Tgn)uaaaauGfc 733AUCACUAUGCAUUUUAAAGUUAA 734 uuaaaguuaaL96 Afuagugsasu AD-392938csusgca(Uhd)UfuUfAfCf 735 asAfsucug(Tgn)acaguaAfa 736GACUGCAUUUUACUGUACAGAUU 737 uguacagauuL96 Afugcagsusc AD-392939ususcug(Chd)UfaUfAfUf 738 usAfsuauc(Agn)caaauaUfa 739GCUUCUGCUAUAUUUGUGAUAUA 740 uugugauauaL96 Gfcagaasgsc AD-392940uscsugc(Uhd)AfuAfUfUf 741 asUfsauau(Cgn)acaaauAfu 742CUUCUGCUAUAUUUGUGAUAUAG 743 ugugauauauL96 Afgcagasasg AD-392941ascsgua(Uhd)CfuUfUfGf 744 asUfscaaa(Ggn)acccaaAfg 745CCACGUAUCUUUGGGUCUUUGAU 746 ggucuuugauL96 Afuacgusgsg AD-392942uscsuuu(Ghd)GfgUfCfUf 747 usCfsuuua(Tgn)caaagaCfc 748UAUCUUUGGGUCUUUGAUAAAGA 749 uugauaaagaL96 Cfaaagasusa AD-392943csusuug(Ghd)GfuCfUfUf 750 usUfscuuu(Agn)ucaaagAfc 751AUCUUUGGGUCUUUGAUAAAGAA 752 ugauaaagaaL96 Cfcaaagsasu AD-392944ususggg(Uhd)CfuUfUfGf 753 usUfsuucu(Tgn)uaucaaAfg 754CUUUGGGUCUUUGAUAAAGAAAA 755 auaaagaaaaL96 Afcccaasasg AD-392945asgsaau(Chd)CfcUfGfUf 756 asUfsuaca(Agn)ugaacaGfg 757AAAGAAUCCCUGUUCAUUGUAAG 758 ucauuguaauL96 Gfauucususu AD-392946gsasauc(Chd)CfuGfUfUf 759 asCfsuuac(Agn)augaacAfg 760AAGAAUCCCUGUUCAUUGUAAGC 761 cauuguaaguL96 Gfgauucsusu AD-392947gsusuca(Uhd)UfgUfAfAf 762 asUfsaaaa(Ggn)ugcuuaCfa 763CUGUUCAUUGUAAGCACUUUUAC 764 gcacuuuuauL96 Afugaacsasg AD-392948ususaug(Ahd)CfaUfGfAf 765 usAfsgaaa(Ggn)cgaucaUfg 766GCUUAUGACAUGAUCGCUUUCUA 767 ucgcuuucuaL96 acauaasgsc AD-392949asusgac(Ahd)UfgAfUfCf 768 usGfsuaga(Agn)agcgauCfa 769UUAUGACAUGAUCGCUUUCUACA 770 gcuuucuacaL96 Ufgucausasa AD-392950csasuga(Uhd)CfgCfUfUf 771 asCfsagug(Tgn)agaaagCfg 772GACAUGAUCGCUUUCUACACUGU 773 ucuacacuguL96 Afucaugsusc AD-392951csusuuc(Uhd)AfcAfCfUf 774 usAfsugua(Agn)uacaguGfu 775CGCUUUCUACACUGUAUUACAUA 776 guauuacauaL96 Afgaaagscsg AD-392952gsasuuc(Ahd)AfuUfUfUf 777 asUfsgguu(Agn)aagaaaAfu 778CAGAUUCAAUUUUCUUUAACCAG 779 cuuuaaccauL96 Ufgaaucsusg AD-392953ususucu(Uhd)UfaAfCfCf 780 asCfsuuca(Ggn)acugguUfa 781AUUUUCUUUAACCAGUCUGAAGU 782 agucugaaguL96 Afagaaasasu AD-392954ususuaa(Ghd)AfuGfUfGf 783 asAfsauug(Agn)agacacAfu 784CUUUUAAGAUGUGUCUUCAAUUu 785 ucuucaauuuL96 Cfuuaaasasg AD-392955ususaag(Ahd)UfgUfGfUf 786 csAfsaauu(Ggn)aagacaCfa 787UUUUAAGAUGUGUCUUCAAUUUG 788 cuucaauuugL96 Ufcuuaasasa AD-392956asgsaug(Uhd)GfuCfUfUf 789 asUfsacaa(Agn)uugaagAfc 790UAAGAUGUGUCUUCAAUUUGUAU 791 caauuuguauL96 Afcaucususa AD-392957usgsucu(Uhd)CfaAfUfUf 792 asUfsuuua(Tgn)acaaauUfg 793UGUGUCUUCAAUUUGUAUAAAAU 794 uguauaaaauL96 Afagacascsa AD-392958csusuca(Ahd)UfuUfGfUf 795 asCfscauu(Tgn)uauacaAfa 796GUCUUCAAUUUGUAUAAAAUGGU 797 auaaaaugguL96 Ufugaagsasc AD-392959asusggu(Ghd)UfuUTUfCf 798 usUfsauuu(Agn)caugaaAfa 799AAAUGGUGUUUUCAUGUAAAUAA 800 auguaaauaaL96 Cfaccaususu AD-392960ususcuu(Uhd)UfaAfGfAf 801 usGfsaaga(Cgn)acaucuUfa 802CCUUCUUUUAAGAUGUGUCUUCA 803 ugugucuucaL96 Afaagaasgsg AD-392961usgsuau(Uhd)CfuAfUfCf 804 usGfsuaaa(Ggn)agagauAfg 805AAUGUAUUCUAUCUCUCUUUACA 806 ucucuuuacaL96 Afauacasusu AD-392962gsuscuc(Uhd)AfuAfCfUf 807 usUfsaaua(Agn)uguaguAfu 808UGGUCUCUAUACUACAUUAUUAA 809 acauuauuaaL96 Afgagacscsa AD-392963uscsucu(Ahd)UfaCfUfAf 810 asUfsuaau(Agn)auguagUfa 811GGUCUCUAUACUACAUUAUUAAU 812 cauuauuaauL96 Ufagagascsc AD-392964csuscua(Uhd)AfcUfAfCf 813 asAfsuuaa(Tgn)aauguaGfu 814GUCUCUAUACUACAUUAUUAAUG 815 auuauuaauuL96 Afuagagsasc AD-392965csusuca(Ahd)UfuAfCfCf 816 asAfsgaau(Tgn)cuugguAfa 817GUCUUCAAUUACCAAGAAUUCUC 818 aagaauucuuL96 Ufugaagsasc AD-392966cscsaca(Chd)AfuCfAfGf 819 asAfsauac(Agn)uuacugAfu 820AUCCACACAUCAGUAAUGUAUUC 821 uaauguauuuL96 Gfuguggsasu AD-392967csusauc(Uhd)CfuCfUfUf 822 asCfsaaaa(Tgn)guaaagAfg 823UUCUAUCUCUCUUUACAUUUUGG 824 uacauuuuguL96 Afgauagsasa AD-392968gsgsucu(Chd)UfaUfAfCf 825 usAfsauaa(Tgn)guaguaUfa 826UUGGUCUCUAUACUACAUUAUUA 827 uacauuauuaL96 Gfagaccsasa AD-392969uscsuau(Ahd)CfuAfCfAf 828 asCfsauua(Agn)uaauguAfg 829UCUCUAUACUACAUUAuuAAUGG 830 uuauuaauguL96 Ufauagasgsa AD-392970gsgsucu(Uhd)CfaAfUfUf 831 asAfsuucu(Tgn)gguaauUfg 832CUGGUCUUCAAUUACCAAGAAUU 833 accaagaauuL96 Afagaccsasg AD-392971csasgga(Uhd)AfuGfAfAf 834 asAfsugau(Ggn)aacuucAfu 835CUCAGGAUAUGAAGUUCAUCAUC 836 guucaucauuL96 Afuccugsasg AD-392972ascsaca(Uhd)CfaGfUfAf 837 usAfsgaau(Agn)cauuacUfg 838CCACACAUCAGUAAUGUAUUCUA 839 auguauucuaL96 Afugugusgsg AD-392973csusaua(Chd)UfaCfAfUf 840 asCfscauu(Agn)auaaugUfa 841CUCUAUACUACAUUAUUAAUGGG 842 uauuaaugguL96 Gfuauagsasg AD-392974cscscgu(Uhd)UfuAfUfGf 843 asUfsgagu(Agn)aaucauAfa 844AACCCGUUUUAUGAUUUACUCAU 845 auuuacucauL96 Afacgggsusu AD-392975ususcca(Uhd)GfaCfUfGf 846 asAfsguaa(Agn)augcagUfc 847UUUUCCAUGACUGCAUUUUACUG 848 cauuuuacuuL96 Afuggaasasa AD-392976uscsuuc(Ahd)AfuUfAfCf 849 asGfsaauu(Cgn)uugguaAfu 850GGUCUUCAAUUACCAAGAAUUCU 851 caagaauucuL96 Ufgaagascsc AD-392977csusgaa(Ghd)UfuUfCfAf 852 asUfsauca(Tgn)aaaugaAfa 853GUCUGAAGUUUCAUUUAUGAUAC 854 uuuaugauauL96 Cfuucagsasc

TABLE 2B Human APP Modified Sequences, No “L96” Linker SEQ SEQ SEQDuplex Sense Sequence ID Antisense Sequence ID ID Name (5′ to 3′) NO(5′ to 3′) NO mRNA target sequence NO AD-392699 gsasccc(Ahd)AfuUfAfAf 33 asAfsagua(Ggn)gacuuaAfu  34 GUGACCCAAUUAAGUCCUACUUU  35 guccuacuuuUfgggucsasc AD-392700 uscsucc(Uhd)GfaUfUfAf  36 asUfsguga(Tgn)aaauaaUfc 37 UCUCUCCUGAUUAUUUAUCACAU  38 uuuaucacau Afggagasgsa AD-392703cscsuga(Ahd)CfuUfGfAf  39 asUfsggau(Tgn)aauucaAfg  40UGCCUGAACUUGAAUUAAUCCAC  41 auuaauccau Ufucaggscsa AD-392704gsgsuuc(Ahd)AfaCfAfAf  42 asAfsuugc(Agn)ccuuugUfu  43UGGGUUCAAACAAAGGUGCAAUC  44 aggugcaauu Ufgaaccscsa AD-392705ususuac(Uhd)CfaUfUfAf  45 csAfsaaag(Ggn)cgauaaUfg  46GAUUUACUCAUUAUCGCCUUUUG  47 ucgccuuuug Afguaaasusc AD-392707asusuua(Ghd)CfuGfUfAf  48 asCfsuagu(Tgn)ugauacAfg  49GAAUUUAGCUGUAUCAAACUAGU  50 ucaaacuagu Cfuaaaususc AD-392708asgsuau(Uhd)CfcUfUfUf  51 asGfsugau(Cgn)aggaaaGfg  52UAAGUAUUCCUUUCCUGAUCACU  53 ccugaucacu Afauacususa AD-392709gscsuua(Uhd)GfaCfAfUf  54 gsAfsaagc(Ggn)aucaugUfc  55UUGCUUAUGACAUGAUCGCUUUC  56 gaucgcuuuc Afuaagcsasa AD-392710asasgau(Ghd)UfgUfCfUf  57 usAfscaaa(Tgn)ugaagaCfa  58UUAAGAUGUGUCUUCAAUUUGUA  59 ucaauuugua Cfaucuusasa AD-392711gscsaaa(Ahd)CfcAfUfUf  60 asufsagug(Agn)agcaauGfg  61CAGCAAAACCAUUGCUUCACUAC  62 gcuucacuau Ufuuugcsusg AD-392712asusuua(Chd)UfcAfUfUf  63 asAfsaagg(Cgn)gauaauGfa  64UGAUUUACUCAUUAUCGCCUUUU  65 aucgccuuuu Gfuaaauscsa AD-392713usascuc(Ahd)UfuAfUfCf  66 asUfscaaa(Agn)ggcgauAfa  67uuuACuCAUUAUCGCCUUUUGAC  68 gccuuuugau Ufgaguasasa AD-392714usgsccu(Ghd)AfaCfUfUf  69 asGfsauua(Agn)uucaagUfu  70GAUGCCUGAACUUGAAUUAAUCC  71 gaauuaaucu Cfaggcasusc AD-392715csusgaa(Chd)UfuGfAfAf  72 usGfsugga(Tgn)uaauucAfa  73GCCUGAACUUGAAUUAAUCCACA  74 uuaauccaca Gfuucagsgsc AD-392716ususuag(Chd)UfgUfAfUf  75 asAfscuag(Tgn)uugauaCfa  76AAUUUAGCUGUAUCAAACuAGuG  77 caaacuaguu Gfcuaaasusu AD-392717gsasaua(Ghd)AfuUfCfUf  78 usAfsauca(Ggn)gagagaAfu  79AuGAAUAGAUUCUCUCCUGAUUA  80 cuccugauua Cfuauucsasu AD-392718uscscug(Ahd)UfuAfUfUf  81 asUfsaugu(Ggn)auaaauAfa  82UCUCCUGAUUAUUUAUCACAUAG  83 uaucacauau Ufcaggasgsa AD-392719cscscaa(Uhd)UfaAfGfUf  84 asUfsaaag(Tgn)aggacuUfa  85GACCCAAUUAAGUCCUACUUUAC  86 ccuacuuuau Afuugggsusc AD-392720csasuau(Ghd)CfuUfUfAf  87 asAfsucga(Tgn)ucuuaaAfg  88UACAUAUGCUUUAAGAAUCGAUG  89 agaaucgauu Cfauaugsusa AD-392721csusucu(Chd)UfuGfCfCf  90 asGfsaaua(Cgn)uuaggcAfa  91UGCUUCUCUUGCCUAAGUAUUCC  92 uaaguauucu Gfagaagscsa AD-392722csasuug(Chd)UfuAfUfGf  93 asCfsgauc(Agn)ugucauAfa  94AUCAUUGCUUAUGACAUGAUCGC  95 acaugaucgu Gfcaaugsasu AD-392723csusuau(Ghd)AfcAfUfGf  96 asGfsaaag(Cgn)gaucauGfu  97UGCUUAUGACAUGAUCGCUUUCU  98 aucgcuuucu Cfauaagscsa AD-392724usasuga(Chd)AfuGfAfUf  99 asufsagaa(Agn)gcgaucAfu 100CUUAUGACAUGAUCGCUUUCUAC 101 cgcuuucuau Gfucauasasg AD-392725usgsaca(Uhd)GfaUfCfGf 102 asufsguag(Agn)aagcgaUfc 103UAUGACAUGAUCGCUUUCUACAC 104 cuuucuacau Afugucasusa AD-392726gsasucg(Chd)UfuUfCfUf 105 asAfsuaca(Ggn)uguaghAfa 106AUGAUCGCUUUCUACACUGUAUU 107 acacuguauu Gfcgaucsasu AD-392727asasaac(Uhd)AfuUfCfAf 108 asGfsacgu(Cgn)aucugaAfu 109GCAAAACUAUUCAGAUGACGUCU 110 gaugacgucu Afguuuusgsc AD-392728asasacu(Ahd)UfuCfAfGf 111 asAfsgacg(Tgn)caucugAfa 112CAAAACUAUUCAGAUGACGUCUU 113 augacgucuu Ufaguuususg AD-392729ascsgaa(Ahd)AfuCfCfAf 114 asCfsuugu(Agn)gguuggAfu 115CUACGAAAAUCCAACCUACAAGU 116 accuacaagu Ufuucgusasg AD-392730usgscuu(Chd)UfcUfUfGf 117 asAfsuacu(Tgn)aggcaaGfa 118GCUGCUUCUCUUGCCUAAGUAUU 119 ccuaaguauu Gfaagcasgsc AD-392731usgscuu(Ahd)UfgAfCfAf 120 asAfsagcg(Agn)ucaughCfa 121AUUGCUUAUGACAUGAUCGCUUU 122 ugaucgcuuu Ufaagcasasu AD-392732usgsauc(Ghd)CfuUfUfCf 123 asUfsacag(Tgn)guagaaAfg 124CAUGAUCGCUUUCUACACUGUAU 125 uacacuguau Cfgaucasusg AD-392733asuscgc(Uhd)UfuCfUfAf 126 usAfsauac(Agn)guguagAfa 127UGAUCGCUUUCUACACUGUAUUA 128 cacuguauua Afgcgauscsa AD-392734uscsuuu(Ghd)AfcCfGfAf 129 asGfsuuuu(Cgn)guuucgGfu 130CAUCUUUGACCGAAACGAAAACC 131 aacgaaaacu Cfaaagasusg AD-392735gsusucu(Ghd)GfgUfUfGf 132 usGfsauau(Tgn)ugucaaCfc 133AGGUUCUGGGUUGACAAAUAUCA 134 acaaauauca Cfagaacscsu AD-392736usgsggu(Uhd)GfaCfAfAf 135 asufscuug(Agn)uauuugUfc 136UCUGGGUUGACAAAUAUCAAGAC 137 auaucaagau Afacccasgsa AD-392737gsasuuu(Ahd)CfuCfAfUf 138 asAfsaggc(Ggn)auaaugAfg 139AUGAUUUACUCAUUAUCGCCUUU 140 uaucgccuuu Ufaaaucsasu AD-392738uscscuu(Uhd)CfcUfGfAf 141 usGfscaua(Ggn)ugaucaGfg 142AUUCCUUUCCUGAUCACUAUGCA 143 ucacuaugca Afaaggasasu AD-392739csusuuc(Chd)UfgAfUfCf 144 asAfsugca(Tgn)agugauCfa 145UCCUUUCCUGAUCACUAUGCAUU 146 acuaugcauu Gfgaaagsgsa AD-392740asusugc(Uhd)UfaUfGfAf 147 asGfscgau(Cgn)augucaUfa 148UCAUUGCUUAUGACAUGAUCGCU 149 caugaucgcu Afgcaausgsa AD-392741uscsuuu(Ahd)AfcCfAfGf 150 asAfsacuu(Cgn)agacugGfh 151UUUCUUUAACCAGUCUGAAGUUU 152 ucugaaguuu Ufaaagasasa AD-392742gsgsauc(Ahd)GfuUfAfCf 153 asAfsucgu(Tgn)uccguaAfc 154AAGGAUCAGUUACGGAAACGAUG 155 ggaaacgauu Ufgauccsusu AD-392743csusggg(Uhd)UfgAfCfAf 156 usCfsuuga(Tgn)auuuguCfa 157UUCUGGGUUGACAAAUAUCAAGA 158 aauaucaaga Afcccagsasa AD-392744asusgau(Uhd)UfaCfUfCf 159 asGfsgcga(Tgn)aaugagUfa 160UUAUGAUUUACUCAUUAUCGCCU 161 auuaucgccu Afaucausasa AD-392745csusugu(Ghd)GfuUTUfGf 162 asAfsuugg(Ggn)ucacaaAfc 163UUCUUGUGGUUUGUGACCCAAUU 164 ugacccaauu Cfacaagsasa AD-392746asusaug(Chd)UfuUfAfAf 165 asCfsaucg(Agn)uucuuaAfa 166ACAUAUGCUUUAAGAAUCGAuGG 167 gaaucgaugu Gfcauausgsu AD-392747ususugu(Chd)CfaCfGfUf 168 asCfsccaa(Agn)gauacgUfg 169UUUUUGUCCACGUAUCUUUGGGU 170 aucuuugggu Gfacaaasasa AD-392748uscsauu(Ghd)UfaAfGfCf 171 asCfsguaa(Agn)agugcuUfa 172GUUCAUUGUAAGCACUUUUACGG 173 acuuuuacgu Cfaaugasasc AD-392749gsgscca(Ahd)CfaUfGfAf 174 asGfsuuca(Cgn)uaaucaUfg 175UUGGCCAACAUGAUUAGUGAACC 176 uuagugaacu Ufuggccsasa AD-392750gsasuca(Ghd)UfuAfCfGf 177 asCfsaucg(Tgn)uuccguAfa 178AGGAUCAGUUACGGAAACGAuGc 179 gaaacgaugu Cfugaucscsu AD-392751usascgg(Ahd)AfaCfGfAf 180 asAfsugag(Agn)gcaucgUfu 181GuuACGGAAACGAUGCUCUCAUG 182 ugcucucauu Ufccguasasc AD-392752usgsauu(Uhd)AfcUfCfAf 183 asAfsggcg(Agn)uaaugaGfu 184UAUGAUUUACUCAUUAUCGCCUU 185 uuaucgccuu Afaaucasusa AD-392753gsusaga(Uhd)GfcCfUfGf 186 asAfsuuca(Agn)guucagGfc 187AAGUAGAUGCCUGAACUUGAAUU 188 aacuugaauu Afucuacsusu AD-392754ususgua(Uhd)AfuUfAfUf 189 asAfsccac(Agn)agaauaAfu 190AGUUGUAUAUUAUUCUUGUGGUU 191 ucuugugguu Afuacaascsu AD-392755asusugc(Uhd)GfcUfUfCf 192 asAfsauau(Agn)gcagaaGfc 193AGAUUGCUGCUUCUGCUAUAUuu 194 ugcuauauuu Afgcaauscsu AD-392756usgscua(Uhd)AfuUfUfGf 195 usCfscuau(Agn)ucacaaAfu 196UCUGCUAUAUUUGUGAUAUAGGA 197 ugauauagga Afuagcasgsa AD-392757ascsaca(Uhd)UfaGfGfCf 198 asAfsgucu(Cgn)aaugccUfa 199GCACACAUUAGGCAUUGAGAcuu 200 auugagacuu Afugugusgsc AD-392758asasgaa(Uhd)CfcCfUfGf 201 usUfsacaa(Tgn)gaacagGfg 202AAAAGAAUCCCUGUUCAUUGUAA 203 uucauuguaa Afuucuususu AD-392759csasuug(Uhd)AfaGfCfAf 204 asCfscgua(Agn)aagugcUfu 205UUCAUUGUAAGCACUUUUACGGG 206 cuuuuacggu Afcaaugsasa AD-392760ususgcu(Uhd)AfuGfAfCf 207 asAfsgcga(Tgn)caugucAfu 208CAUUGCUUAUGACAUGAUCGCUU 209 augaucgcuu Afagcaasusg AD-392761csasagg(Ahd)UfcAfGfUf 210 asGfsuuuc(Cgn)guaacuGfa 211ACCAAGGAUCAGUUACGGAAACG 212 uacggaaacu Ufccuugsgsu AD-392762asgsguu(Chd)UfgGfGfUf 213 asUfsauuu(Ggn)ucaaccCfa 214CCAGGUUCUGGGUUGACAAAUAU 215 ugacaaauau Gfaaccusgsg AD-392763asasgau(Ghd)UfgGfGfUf 216 asUfsuugu(Tgn)ugaaccCfa 217AGAAGAUGUGGGUUCAAACAAAG 218 ucaaacaaau Cfaucuuscsu AD-392764csusgaa(Ghd)AfaGfAfAf 219 usGfsugua(Cgn)uguuucUfu 220UGCUGAAGAAGAAACAGUACACA 221 acaguacaca Cfuucagscsa AD-392765asasguu(Ghd)GfaCfAfGf 222 asAfsuggu(Tgn)uugcugUfc 223UGAAGUUGGACAGCAAAACCAUU 224 caaaaccauu Cfaacuuscsa AD-392766asuscgg(Uhd)GfuCfCfAf 225 asUfsucua(Tgn)aaauggAfc 226CCAUCGGUGUCCAUUUAUAGAAU 227 uuuauagaau Afccgausgsg AD-392767uscsggu(Ghd)UfcCfAfUf 228 usAfsuucu(Agn)uaaaugGfa 229CAUCGGUGUCCAUUUAUAGAAUA 230 uuauagaaua Cfaccgasusg AD-392768gscsugu(Ahd)AfcAfCfAf 231 asGfscauc(Tgn)acuuguGfu 232GUGCUGUAACACAAGUAGAUGCC 233 aguagaugcu Ufacagcsasc AD-392769asasgua(Ghd)AfuGfCfCf 234 usUfscaag(Tgn)ucaggcAfu 235ACAAGUAGAUGCCUGAACUUGAA 236 ugaacuugaa Cfuacuusgsu AD-392770ususgug(Ghd)UfuUfGfUf 237 usAfsauug(Ggn)gucacaAfa 238UCUUGUGGUUUGUGACCCAAUUA 239 gacccaauua Cfcacaasgsa AD-392771gsusuug(Uhd)GfaCfCfCf 240 asGfsacuu(Agn)auugggUfc 241UGGUUUGUGACCCAAUUAAGUCC 242 aauuaagucu Afcaaacscsa AD-392772gsusgac(Chd)CfaAfUfUf 243 asGfsuagg(Agn)cuuaauUfg 244UUGUGACCCAAUUAAGUCCUACU 245 aaguccuacu Gfgucacsasa AD-392773usasugc(Uhd)UfuAfAfGf 246 asCfscauc(Ggn)auucuuAfa 247CAUAUGCUUUAAGAAUCGAUGGG 248 aaucgauggu Afgcauasusg AD-392774ususugu(Ghd)AfuAfUfAf 249 usCfsuuaa(Tgn)uccuauAfu 250UAUUUGUGAUAUAGGAAUUAAGA 251 ggaauuaaga Cfacaaasusa AD-392775asasaga(Ahd)UfcCfCfUf 252 usAfscaau(Ggn)aacaggGfa 253GAAAAGAAUCCCUGUUCAUUGUA 254 guucauugua Ufucuuususc AD-392776usgsauu(Ghd)UfaCfAfGf 255 asGfscaau(Ggn)auucugUfa 256GAUGAUUGUACAGAAUCAUUGCU 257 aaucauugcu Cfaaucasusc AD-392777usgsccu(Ghd)GfaCfAfAf 258 asAfsagaa(Ggn)gguuugUfc 259CAUGCCUGGACAAACCCUUCUUU 260 acccuucuuu Cfaggcasusg AD-392778gsasgca(Ahd)AfaCfUfAf 261 asUfscauc(Tgn)gaauagUfu 262AAGAGCAAAACUAUUCAGAUGAC 263 uucagaugau Ufugcucsusu AD-392779asgsuga(Ahd)CfcAfAfGf 264 asUfsaacu(Ggn)auccuuGfg 265UUAGUGAACCAAGGAUCAGUUAC 266 gaucaguuau Ufucacusasa AD-392780usgsaac(Chd)AfaGfGfAf 267 asCfsguaa(Cgn)ugauccUfu 268AGUGAACCAAGGAUCAGUUAcGG 269 ucaguuacgu Gfguucascsu AD-392781csasguu(Ahd)CfgGfAfAf 270 asGfsagca(Tgn)cguuucCfg 271AUCAGUUACGGAAACGAUGCUCU 272 acgaugcucu Ufaacugsasu AD-392782asgsaag(Ahd)UfgUfGfGf 273 usUfsguuu(Ggn)aacccaCfa 274GCAGAAGAUGUGGGUUCAAACAA 275 guucaaacaa Ufcuucusgsc AD-392783cscsucu(Ghd)AfaGfUfUf 276 usUfsugcu(Ggn)uccaacUfu 277AGCCUCUGAAGUUGGACAGCAAA 278 ggacagcaaa Cfagaggscsu AD-392784ususaug(Ahd)UfuUfAfCf 279 ascfsgaua(Agn)ugaguaAfa 280UUUUAUGAUUUACUCAUUAUCGC 281 ucauuaucgu Ufcauaasasa AD-392785ascsagc(Uhd)GfuGfCfUf 282 asUfsugug(Tgn)uacagcAfc 283UGACAGCUGUGCUGUAACACAAG 284 guaacacaau Afgcuguscsa AD-392786usgsuga(Chd)CfcAfAfUf 285 asufsagga(Cgn)uuaauuGfg 286UUUGUGACCCAAUUAAGUCCUAC 287 uaaguccuau Gfucacasasa AD-392787usascau(Ahd)UfgCfUfUf 288 usCfsgauu(Cgn)uuaaagCfa 289UUUACAUAUGCUUUAAGAAUCGA 290 uaagaaucga Ufauguasasa AD-392788gsusaaa(Uhd)AfaAfUfAf 291 usCfscaag(Agn)auguauUfu 292AUGUAAAUAAAUACAUUCUUGGA 293 cauucuugga Afuuuacsasu AD-392789uscsagu(Uhd)AfcGfGfAf 294 asAfsgcau(Cgn)guuuccGfu 295GAUCAGUUACGGAAACGAUGCUC 296 aacgaugcuu Afacugasusc AD-392790csusucc(Chd)GfuGfAfAf 297 asAfsacuc(Tgn)ccauucAfc 298UCCUUCCCGUGAAUGGAGAGUUC 299 uggagaguuu Gfggaagsgsa AD-392791asgsuug(Ghd)AfcAfGfCf 300 asAfsaugg(Tgn)uuugcuGfu 301GAAGUUGGACAGCAAAACCAUUG 302 aaaaccauuu Cfcaacususc AD-392792cscscau(Chd)GfgUfGfUf 303 asUfsauaa(Agn)uggacaCfc 304UACCCAUCGGUGUCCAUUUAUAG 305 ccauuuauau Gfaugggsusa AD-392793usgscac(Ahd)CfaUfUfAf 306 usCfsucaa(Tgn)gccuaaUfg 307UGUGCACACAUUAGGCAUUGAGA 308 ggcauugaga Ufgugcascsa AD-392794cscsaac(Ahd)UfgAfUfUf 309 usufsgguu(Cgn)acuaauCfa 310GGCCAACAUGAUUAGUGAACCAA 311 agugaaccaa Ufguuggscsc AD-392795asusgau(Uhd)AfgUfGfAf 312 asAfsuccu(Tgn)gguucaCfu 313ACAUGAUUAGUGAACCAAGGAUC 314 accaaggauu Afaucausgsu AD-392796ususagu(Ghd)AfaCfCfAf 315 asAfscuga(Tgn)ccuuggUfu 316GAUUAGUGAACCAAGGAUCAGUU 317 aggaucaguu Cfacuaasusc AD-392797asascca(Ahd)GfgAfUfCf 318 usUfsccgu(Agn)acugauCfc 319uGAACCAAGGAUCAGUUACGGAA 320 aguuacggaa Ufugguuscsa AD-392798gsusuac(Ghd)GfaAfAfCf 321 usGfsagag(Cgn)aucguuUfc 322CAGUUACGGAAACGAUGCUCUCA 323 gaugcucuca Cfguaacsusg AD-392799gsasugc(Ahd)GfaAfUfUf 324 asUfscaug(Tgn)cggaauUfc 325uGGAUGCAGAAUUCCGACAUGAC 326 ccgacaugau Ufgcaucscsa AD-392800ususgga(Chd)AfgCfAfAf 327 asGfscaau(Ggn)guuuugCfu 328AGUUGGACAGCAAAACCAUUGCU 329 aaccauugcu Gfuccaascsu AD-392801asasacc(Ahd)UfuGfCfUf 330 usGfsggua(Ggn)ugaagcAfa 331CAAAACCAUUGCUUCACUACCCA 332 ucacuaccca Ufgguuususg AD-392802cscsauc(Ghd)GfuGfUfCf 333 usCfsuaua(Agn)auggacAfc 334ACCCAUCGGUGUCCAUUUAUAGA 335 cauuuauaga Cfgauggsgsu AD-392803ususauc(Ghd)CfcUfUfUf 336 asCfsagcu(Ggn)ucaaaaGfg 337CAUUAUCGCCUUUUGACAGCUGU 338 ugacagcugu Cfgauaasusg AD-392804asuscgc(Chd)UfuUfUfGf 339 asCfsacag(Cgn)ugucaaAfa 340UUAUCGCCUUUUGACAGCUGUGC 341 acagcugugu Gfgcgausasa AD-392805ascsaca(Ahd)GfuAfGfAf 342 asGfsuuca(Ggn)gcaucuAfc 343UAACACAAGUAGAUGCCUGAACU 344 ugccugaacu Ufugugususa AD-392806usgsugg(Uhd)UfuGfUfGf 345 usufsaauu(Ggn)ggucacAfa 346CUUGUGGUUUGUGACCCAAUUAA 347 acccaauuaa Afccacasasg AD-392807gsgsgau(Ghd)CfuUfCfAf 348 asAfscguu(Cgn)acaugaAfg 349GGGGGAUGCUUCAUGUGAACGUG 350 ugugaacguu Cfaucccscsc AD-392808usgsugc(Ahd)CfaCfAfUf 351 usCfsaaug(Cgn)cuaaugUfg 352UAUGUGCACACAUUAGGCAUUGA 353 uaggcauuga Ufgcacasusa AD-392809asasaug(Ghd)AfaGfUfGf 354 asUfsuaua(Tgn)ugccacUfu 355GAAAAUGGAAGUGGCAAUAUAAG 356 gcaauauaau Cfcauuususc AD-392810asusgga(Ahd)GfuGfGfCf 357 asCfscuua(Tgn)auugccAfc 358AAAUGGAAGUGGCAAUAUAAGGG 359 aauauaaggu Ufuccaususu AD-392811usgsccc(Ghd)AfgAfUfCf 360 asGfsuuua(Agn)caggauCfu 361CUUGCCCGAGAUCCUGUUAAACU 362 cuguuaaacu Cfgggcasasg AD-392812asusuag(Uhd)GfaAfCfCf 363 asCfsugau(Cgn)cuugguUfc 364UGAUUAGUGAACCAAGGAUCAGU 365 aaggaucagu Afcuaauscsa AD-392813gsasacc(Ahd)AfgGfAfUf 366 usCfscgua(Agn)cugaucCfu 367GUGAACCAAGGAUCAGUUACGGA 368 caguuacgga Ufgguucsasc AD-392814asasgga(Uhd)CfaGfUfUf 369 usCfsguuu(Cgn)cguaacUfg 370CCAAGGAUCAGUUACGGAAACGA 371 acggaaacga Afuccuusgsg AD-392815csasaca(Chd)AfgAfAfAf 372 usCfsaacu(Tgn)cguuuuCfu 373GCCAACACAGAAAACGAAGUUGA 374 acgaaguuga Gfuguugsgsc AD-392816usgsggu(Uhd)CfaAfAfCf 375 usUfsgcac(Cgn)uuuguuUfg 376UGUGGGUUCAAACAAAGGUGCAA 377 aaaggugcaa Afacccascsa AD-392817csasgug(Ahd)UfcGfUfCf 378 ascfsaagg(Tgn)gaugacGfa 379GACAGUGAUCGUCAUCACCUUGG 380 aucaccuugu Ufcacugsusc AD-392818ascscca(Uhd)CfgGfUfGf 381 usAfsuaaa(Tgn)ggacacCfg 382CUACCCAUCGGUGUCCAUUUAUA 383 uccauuuaua Afugggusasg AD-392819uscsuug(Uhd)GfgUfUfUf 384 asUfsuggg(Tgn)cachaaCfc 385AUUCUUGUGGUUUGUGACCCAAU 386 gugacccaau Afcaagasasu AD-392820ususugu(Ghd)AfcCfCfAf 387 asGfsgacu(Tgn)aauuggGfu 388GGUUUGUGACCCAAUUAAGUCCU 389 auuaaguccu Cfacaaascsc AD-392821ususgug(Ahd)CfcCfAfAf 390 usAfsggac(Tgn)uhahugGfg 391GUUUGUGACCCAAUUAAGUCCUA 392 uuaaguccua Ufcachasasc AD-392822ususcag(Ahd)UfgAfCfGf 393 usUfsggcc(Agn)agacguCfa 394UAUUCAGAUGACGUCUUGGCCAA 395 ucuuggccaa Ufcugaasusa AD-392823asuscag(Uhd)UfaCfGfGf 396 asGfscauc(Ggn)uuuccgUfa 397GGAUCAGUUACGGAAACGAUGCU 398 aaacgaugcu Afcugauscsc AD-392824usgsgau(Ghd)CfaGfAfAf 399 asAfsuguc(Ggn)gaauucUfg 400GAUGGAUGCAGAAUUCCGACAUG 401 uuccgacauu Cfauccasusc AD-392825gsuscca(Ahd)GfaUfGfCf 402 asCfsguuc(Tgn)gcugcaUfc 403CUGUCCAAGAUGCAGCAGAACGG 404 agcagaacgu Ufuggacsasg AD-392826usasccc(Ahd)UfcGfGfUf 405 asUfsaaau(Ggn)gacaccGfa 406ACUACCCAUCGGUGUCCAUUUAU 407 guccauuuau Ufggguasgsu AD-392827ususuug(Ahd)CfaGfCfUf 408 asUfsuaca(Ggn)cacagcUfg 409CCUUUUGACAGCUGUGCUGUAAC 410 gugcuguaau Ufcaaaasgsg AD-392828ususgac(Ahd)GfcUfGfUf 411 asUfsgtma(Cgn)agcacaGfc 412UUUUGACAGCUGUGCUGUAACAC 413 gcuguaacau Ufgucaasasa AD-392829asgscug(Uhd)GfcUfGfUf 414 usAfscuug(Tgn)guuacaGfc 415ACAGCUGUGCUGUAACACAAGUA 416 aacacaagua Afcagcusgsu AD-392830gsusuuu(Ahd)UfgUfGfCf 417 asCfsuaau(Ggn)ugugcaCfa 418CUGUUUUAUGUGCACACAUUAGG 419 acacauuagu Ufaaaacsasg AD-392831ususcaa(Uhd)UfaCfCfAf 420 usGfsagaa(Tgn)ucuuggUfa 421UCUUCAAUUACCAAGAAUUCUCC 422 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AD-392930usasagu(Chd)CfuAfCfUf 711 asCfsauau(Ggn)uaaaguAfg 712AUUAAGUCCUACUUUACAUAUGC 713 uuacauaugu Gfacuuasasu AD-392931asgsucc(Uhd)AfcUfUfUf 714 asAfsgcau(Agn)uguaaaGfu 715UAAGUCCUACUUUACAUAUGCUU 716 acauaugcuu Afggacususa AD-392932gsusccu(Ahd)CfuUTUfAf 717 asAfsagca(Tgn)auguaaAfg 718AAGUCCUACUUUACAUAUGCUUU 719 cauaugcuuu Ufaggacsusu AD-392933ususcuc(Uhd)UfgCfCfUf 720 asGfsgaau(Agn)cuuaggCfa 721GCUUCUCUUGCCUAAGUAUUCCU 722 aaguauuccu Afgagaasgsc AD-392934csuscuu(Ghd)CfcUfAfAf 723 asAfsagga(Agn)uacuuaGfg 724UUCUCUUGCCUAAGUAUUCCUUU 725 guauuccuuu Cfaagagsasa AD-392935usasuuc(Chd)UfuUfCfCf 726 asUfsagug(Agn)ucaggaAfa 727AGUAUUCCUUUCCUGAUCACUAU 728 ugaucacuau Gfgaauascsu AD-392936ususucc(Uhd)GfaUfCfAf 729 asAfsaugc(Agn)uagugaUfc 730CCUUUCCUGAUCACUAUGCAUUU 731 cuaugcauuu Afggaaasgsg AD-392937csascua(Uhd)GfcAfUfUf 732 usufsaacu(Tgn)uaaaauGfc 733AUCACUAUGCAUUUUAAAGUUAA 734 uuaaaguuaa Afuagugsasu AD-392938csusgca(Uhd)UfuUfAfCf 735 asAfsucug(Tgn)acaguaAfa 736GACUGCAUUUUACUGUACAGAUU 737 uguacagauu Afugcagsusc AD-392939ususcug(Chd)UfaUfAfUf 738 usAfsuauc(Agn)caaauaUfa 739GCUUCUGCUAUAUUUGUGAUAUA 740 uugugauaua Gfcagaasgsc AD-392940uscsugc(Uhd)AfuAfUfUf 741 asUfsauau(Cgn)acaaauAfu 742CUUCUGCUAUAUUUGUGAUAUAG 743 ugugauauau Afgcagasasg AD-392941ascsgua(Uhd)CfuUTUfGf 744 asUfscaaa(Ggn)acccaaAfg 745CCACGUAUCUUUGGGUCUUUGAU 746 ggucuuugau Afuacgusgsg AD-392942uscsuuu(Ghd)GfgUfCfUf 747 uscfsuuua(Tgn)caaagaCfc 748UAUCUUUGGGUCUUUGAUAAAGA 749 uugauaaaga Cfaaagasusa AD-392943csusuug(Ghd)GfuCfUfUf 750 usufscuuu(Agn)uchaagAfc 751AUCUUUGGGUCUUUGAUAAAGAA 752 ugauaaagaa Cfcaaagsasu AD-392944ususggg(Uhd)CfuUTUfGf 753 usUfsuucu(Tgn)uaucaaAfg 754CUUUGGGUCUUUGAUAAAGAAAA 755 auaaagaaaa Afcccaasasg AD-392945asgsaau(Chd)CfcUfGfUf 756 asUfsuaca(Agn)ugaacaGfg 757AAAGAAUCCCUGUUCAUUGUAAG 758 ucauuguaau Gfauucususu AD-392946gsasauc(Chd)CfuGfUfUf 759 asCfsuuac(Agn)augaacAfg 760AAGAAUCCCUGUUCAUUGUAAGC 761 cauuguaagu Gfgauucsusu AD-392947gsusuca(Uhd)UfgUfAfAf 762 asUfsaaaa(Ggn)ugcuuaCfa 763CUGUUCAUUGUAAGCACUUUUAC 764 gcacummau Afugaacsasg AD-392948ususaug(Ahd)CfaUfGfAf 765 usAfsgaaa(Ggn)cgaucaUfg 766GCUUAUGACAUGAUCGCUUUCUA 767 ucgcuuucua Ufcauaasgsc AD-392949asusgac(Ahd)UfgAfUfCf 768 usGfsuaga(Agn)agcgauCfa 769UUAUGACAUGAUCGCUUUCUACA 770 gcuuucuaca Ufgucausasa AD-392950csasuga(Uhd)CfgCfUfUf 771 asCfsagug(Tgn)agaaagCfg 772GACAUGAUCGCUUUCUACACUGU 773 ucuacacugu Afucaugsusc AD-392951csusuuc(Uhd)AfcAfCfUf 774 usAfsugua(Agn)uacaguGfu 775CGCUUUCUACACUGUAUUACAUA 776 guauuacaua Afgaaagscsg AD-392952gsasuuc(Ahd)AfuUfUfUf 777 asUfsgguu(Agn)aagaaaAfu 778CAGAUUCAAUUUUCUUUAACCAG 779 cuuuaaccau Ufgaaucsusg AD-392953ususucu(Uhd)UfaAfCfCf 780 asCfsuuca(Ggn)acugguUfa 781AUUUUCUUUAACCAGUCUGAAGU 782 agucugaagu Afagaaasasu AD-392954ususuaa(Ghd)AfuGfUfGf 783 asAfsauug(Agn)agacacAfu 784CUUUUAAGAUGUGUCUUCAAUUU 785 ucuucaauuu Cfuuaaasasg AD-392955ususaag(Ahd)UfgUfGfUf 786 csAfsaauu(Ggn)aagacaCfa 787UUUUAAGAUGUGUCUUCAAUUUG 788 cuucaauuug Ufcuuaasasa AD-392956asgsaug(Uhd)GfuCfUfUf 789 asUfsacaa(Agn)uugaagAfc 790UAAGAUGUGUCUUCAAUUUGUAU 791 caauuuguau Afcaucususa AD-392957usgsucu(Uhd)CfaAfUfUf 792 asufsuuua(Tgn)acaaauUfg 793UGUGUCUUCAAUUUGUAUAAAAU 794 uguauaaaau Afagacascsa AD-392958csusuca(Ahd)UfuUfGfUf 795 asCfscauu(Tgn)uauacaAfa 796GUCUUCAAUUUGUAUAAAAUGGU 797 auaaaauggu Ufugaagsasc AD-392959asusggu(Ghd)UfuUfUfCf 798 usUfsauuu(Agn)caugaaAfa 799AAAUGGUGUUUUCAUGUAAAUAA 800 auguaaauaa Cfaccaususu AD-392960ususcuu(Uhd)UfaAfGfAf 801 usGfsaaga(Cgn)acaucuUfa 802CCUUCUUUUAAGAUGUGUCUUCA 803 ugugucuuca Afaagaasgsg AD-392961usgsuau(Uhd)CfuAfUfCf 804 usGfsuaaa(Ggn)agagahAfg 805AAUGUAUUCUAUCUCUCUUUACA 806 ucucuuuaca Afauacasusu AD-392962gsuscuc(Uhd)AfuAfCfUf 807 usUfsaaua(Agn)uguaguAfu 808UGGUCUCUAUACUACAUUAUUAA 809 acauuauuaa Afgagacscsa AD-392963uscsucu(Ahd)UfaCfUfAf 810 asUfsuaau(Agn)auguagUfa 811GGUCUCUAUACUACAUUAUUAAU 812 cauuuuuaau Ufagagascsc AD-392964csuscua(Uhd)AfcUfAfCf 813 asAfsuuaa(Tgn)aauguaGfu 814GUCUCUAUACUACAUUAUUAAUG 815 auuauuaauu Afuagagsasc AD-392965csusuca(Ahd)UfuAfCfCf 816 asAfsgaau(Tgn)cuugguAfa 817GUCUUCAAUUACCAAGAAUUCUC 818 aagaauucuu Ufugaagsasc AD-392966cscsaca(Chd)AfuCfAfGf 819 asAfsauac(Agn)uuacugAfu 820AUCCACACAUCAGUAAUGUAUUC 821 uaauguauuu Gfuguggsasu AD-392967csusauc(Uhd)CfuCfUfUf 822 asCfsaaaa(Tgn)guaaagAfg 823UUCUAUCUCUCUUUACAUUUUGG 824 uacauuuugu Afgauagsasa AD-392968gsgsucu(Chd)UfaUfAfCf 825 usAfsauaa(Tgn)guaguaUfa 826UUGGUCUCUAUACUACAUUAUUA 827 uacauuauua Gfagaccsasa AD-392969uscsuau(Ahd)CfuAfCfAf 828 asCfsauua(Agn)uaauguAfg 829UCUCUAUACUACAUUAuuAAuGG 830 uuauuaaugu Ufauagasgsa AD-392970gsgsucu(Uhd)CfaAfUfUf 831 asAfsuucu(Tgn)gguaauUfg 832CUGGUCUUCAAUUACCAAGAAUU 833 accaagaauu Afagaccsasg AD-392971csasgga(Uhd)AfuGfAfAf 834 asAfsugau(Ggn)aacuucAfu 835CuCAGGAUAUGAAGUUCAUCAUC 836 guucaucauu Afuccugsasg AD-392972ascsaca(Uhd)CfaGfUfAf 837 usAfsgaau(Agn)cauuacUfg 838CCACACAUCAGUAAUGUAUUCUA 839 auguauucua Afugugusgsg AD-392973csusaua(Chd)UfaCfAfUf 840 asCfscauu(Agn)auaaugUfa 841CUCUAUACUACAUUAUUAAUGGG 842 uauuaauggu Gfuauagsasg AD-392974cscscgu(Uhd)UfuAfUfGf 843 asUfsgagu(Agn)aaucauAfa 844AACCCGUUUUAUGAUUUACUCAU 845 auuuacucau Afacgggsusu AD-392975ususcca(Uhd)GfaCfUfGf 846 asAfsguaa(Agn)augcagUfc 847UUUUCCAUGACUGCAUUUUACUG 848 cauuuuacuu Afuggaasasa AD-392976uscsuuc(Ahd)AfuUfAfCf 849 asGfsaauu(Cgn)uugguaAfu 850GGUCUUCAAUUACCAAGAAUUCU 851 caagaauucu Ufgaagascsc AD-392977csusgaa(Ghd)UfuUTCfAf 852 asUfsauca(Tgn)aaaugaAfa 853GUCUGAAGUUUCAUUUAUGAUAC 854 uuuaugauau Cfuucagsasc

TABLE 3 APP Unmodified Sequences, Human NM_000484 Targeting SEQ PositionSEQ Position Sense Sequence ID in Antisense Sequence ID in Duplex Name(5′ to 3′) NO NM_000484 (5′ to 3′) NO NM_000484 AD-392853GCGCCAUGUCCCAAAGUUUAU  855 1228-1248 AUAAACTUUGGGACAUGGCGCUG  8561226-1248 AD-392857 CUUGCCCGAGAUCCUGUUAAA  857 1269-1289UUUAACAGGAUCUCGGGCAAGAG  858 1267-1289 AD-392851 UUGCCCGAGAUCCUGUUAAAU 859 1270-1290 AUUUAACAGGAUCUCGGGCAAGA  860 1268-1290 AD-392811UGCCCGAGAUCCUGUUAAACU  861 1271-1291 AGUUUAACAGGAUCUCGGGCAAG  8621269-1291 AD-392910 GAUCCUGUUAAACUUCCUACA  863 1278-1298UGUAGGAAGUUUAACAGGAUCUC  864 1276-1298 AD-392890 AUCCUGUUAAACUUCCUACAA 865 1279-1299 UUGUAGGAAGUUUAACAGGAUCU  866 1277-1299 AD-392911CUGCUUCAGAAAGAGCAAAAU  867 1893-1913 AUUUUGCUCUUUCUGAAGCAGCU  8681891-1913 AD-392912 CAGAAAGAGCAAAACUAUUCA  869 1899-1919UGAAUAGUUUUGCUCUUUCUGAA  870 1897-1919 AD-392778 GAGCAAAACUAUUCAGAUGAU 871 1905-1925 AUCAUCTGAAUAGUUUUGCUCUU  872 1903-1925 AD-392727AAAACUAUUCAGAUGACGUCU  873 1909-1929 AGACGUCAUCUGAAUAGUUUUGC  8741907-1929 AD-392728 AAACUAUUCAGAUGACGUCUU  875 1910-1930AAGACGTCAUCUGAAUAGUUUUG  876 1908-1930 AD-392891 ACUAUUCAGAUGACGUCUUGU 877 1912-1932 ACAAGACGUCAUCUGAAUAGUUU  878 1910-1932 AD-392822UUCAGAUGACGUCUUGGCCAA  879 1916-1936 UUGGCCAAGACGUCAUCUGAAUA  8801914-1936 AD-392749 GGCCAACAUGAUUAGUGAACU  881 1931-1951AGUUCACUAAUCAUGUUGGCCAA  882 1929-1951 AD-392794 CCAACAUGAUUAGUGAACCAA 883 1933-1953 UUGGUUCACUAAUCAUGUUGGCC  884 1931-1953 AD-392795AUGAUUAGUGAACCAAGGAUU  885 1938-1958 AAUCCUTGGUUCACUAAUCAUGU  8861936-1958 AD-392812 AUUAGUGAACCAAGGAUCAGU  887 1941-1961ACUGAUCCUUGGUUCACUAAUCA  888 1939-1961 AD-392796 UUAGUGAACCAAGGAUCAGUU 889 1942-1962 AACUGATCCUUGGUUCACUAAUC  890 1940-1962 AD-392779AGUGAACCAAGGAUCAGUUAU  891 1944-1964 AUAACUGAUCCUUGGUUCACUAA  8921942-1964 AD-392780 UGAACCAAGGAUCAGUUACGU  893 1946-1966ACGUAACUGAUCCUUGGUUCACU  894 1944-1966 AD-392813 GAACCAAGGAUCAGUUACGGA 895 1947-1967 UCCGUAACUGAUCCUUGGUUCAC  896 1945-1967 AD-392797AACCAAGGAUCAGUUACGGAA  897 1948-1968 UUCCGUAACUGAUCCUUGGUUCA  8981946-1968 AD-392761 CAAGGAUCAGUUACGGAAACU  899 1951-1971AGUUUCCGUAACUGAUCCUUGGU  900 1949-1971 AD-392814 AAGGAUCAGUUACGGAAACGA 901 1952-1972 UCGUUUCCGUAACUGAUCCUUGG  902 1950-1972 AD-392742GGAUCAGUUACGGAAACGAUU  903 1954-1974 AAUCGUTUCCGUAACUGAUCCUU  9041952-1974 AD-392750 GAUCAGUUACGGAAACGAUGU  905 1955-1975ACAUCGTUUCCGUAACUGAUCCU  906 1953-1975 AD-392823 AUCAGUUACGGAAACGAUGCU 907 1956-1976 AGCAUCGUUUCCGUAACUGAUCC  908 1954-1976 AD-392789UCAGUUACGGAAACGAUGCUU  909 1957-1977 AAGCAUCGUUUCCGUAACUGAUC  9101955-1977 AD-392781 CAGUUACGGAAACGAUGCUCU  911 1958-1978AGAGCATCGUUUCCGUAACUGAU  912 1956-1978 AD-392798 GUUACGGAAACGAUGCUCUCA 913 1960-1980 UGAGAGCAUCGUUUCCGUAACUG  914 1958-1980 AD-392751UACGGAAACGAUGCUCUCAUU  915 1962-1982 AAUGAGAGCAUCGUUUCCGUAAC  9161960-1982 AD-392858 CUCAUGCCAUCUUUGACCGAA  917 1977-1997UUCGGUCAAAGAUGGCAUGAGAG  918 1975-1997 AD-392844 UCAUGCCAUCUUUGACCGAAA 919 1978-1998 UUUCGGTCAAAGAUGGCAUGAGA  920 1976-1998 AD-392842CAUGCCAUCUUUGACCGAAAU  921 1979-1999 AUUUCGGUCAAAGAUGGCAUGAG  9221977-1999 AD-392848 AUGCCAUCUUUGACCGAAACU  923 1980-2000AGUUUCGGUCAAAGAUGGCAUGA  924 1978-2000 AD-392838 GCCAUCUUUGACCGAAACGAA 925 1982-2002 UUCGUUTCGGUCAAAGAUGGCAU  926 1980-2002 AD-392839CCAUCUUUGACCGAAACGAAA  927 1983-2003 UUUCGUTUCGGUCAAAGAUGGCA  9281981-2003 AD-392734 UCUUUGACCGAAACGAAAACU  929 1986-2006AGUUUUCGUUUCGGUCAAAGAUG  930 1984-2006 AD-392790 CUUCCCGUGAAUGGAGAGUUU 931 2019-2039 AAACUCTCCAUUCACGGGAAGGA  932 2017-2039 AD-392815CAACACAGAAAACGAAGUUGA  933 2093-2113 UCAACUTCGUUUUCUGUGUUGGC  9342091-2113 AD-392762 AGGUUCUGGGUUGACAAAUAU  935 2162-2182AUAUUUGUCAACCCAGAACCUGG  936 2160-2182 AD-392735 GUUCUGGGUUGACAAAUAUCA 937 2164-2184 UGAUAUTUGUCAACCCAGAACCU  938 2162-2184 AD-392743CUGGGUUGACAAAUAUCAAGA  939 2167-2187 UCUUGATAUUUGUCAACCCAGAA  9402165-2187 AD-392736 UGGGUUGACAAAUAUCAAGAU  941 2168-2188AUCUUGAUAUUUGUCAACCCAGA  942 2166-2188 AD-392824 UGGAUGCAGAAUUCCGACAUU 943 2212-2232 AAUGUCGGAAUUCUGCAUCCAUC  944 2210-2232 AD-392799GAUGCAGAAUUCCGACAUGAU  945 2214-2234 AUCAUGTCGGAAUUCUGCAUCCA  9462212-2234 AD-392971 CAGGAUAUGAAGUUCAUCAUU  947 2236-2256AAUGAUGAACUUCAUAUCCUGAG  948 2234-2256 AD-392913 UAUGAAGUUCAUCAUCAAAAA 949 2241-2261 UUUUUGAUGAUGAACUUCAUAUC  950 2239-2261 AD-392892GUUCAUCAUCAAAAAUUGGUU  951 2247-2267 AACCAATUUUUGAUGAUGAACUU  9522245-2267 AD-392914 CAUCAUCAAAAAUUGGUGUUU  953 2250-2270AAACACCAAUUUUUGAUGAUGAA  954 2248-2270 AD-392860 CAUCAAAAAUUGGUGUUCUUU 955 2253-2273 AAAGAACACCAAUUUUUGAUGAU  956 2251-2273 AD-392875AUCAAAAAUUGGUGUUCUUUG  957 2254-2274 CAAAGAACACCAAUUUUUGAUGA  9582252-2274 AD-392915 UCAAAAAUUGGUGUUCUUUGU  959 2255-2275ACAAAGAACACCAAUUUUUGAUG  960 2253-2275 AD-392782 AGAAGAUGUGGGUUCAAACAA 961 2276-2296 UUGUUUGAACCCACAUCUUCUGC  962 2274-2296 AD-392763AAGAUGUGGGUUCAAACAAAU  963 2278-2298 AUUUGUTUGAACCCACAUCUUCU  9642276-2298 AD-392816 UGGGUUCAAACAAAGGUGCAA  965 2284-2304UUGCACCUUUGUUUGAACCCACA  966 2282-2304 AD-392704 GGUUCAAACAAAGGUGCAAUU 967 2286-2306 AAUUGCACCUUUGUUUGAACCCA  968 2284-2306 AD-392854GUCAUAGCGACAGUGAUCGUU  969 2331-2351 AACGAUCACUGUCGCUAUGACAA  9702329-2351 AD-392856 AUAGCGACAGUGAUCGUCAUU  971 2334-2354AAUGACGAUCACUGUCGCUAUGA  972 2332-2354 AD-392817 CAGUGAUCGUCAUCACCUUGU 973 2341-2361 ACAAGGTGAUGACGAUCACUGUC  974 2339-2361 AD-392764CUGAAGAAGAAACAGUACACA  975 2367-2387 UGUGUACUGUUUCUUCUUCAGCA  9762365-2387 AD-392845 CAGUACACAUCCAUUCAUCAU  977 2379-2399AUGAUGAAUGGAUGUGUACUGUU  978 2377-2399 AD-392825 GUCCAAGAUGCAGCAGAACGU 979 2447-2467 ACGUUCTGCUGCAUCUUGGACAG  980 2445-2467 AD-392849GAACGGCUACGAAAAUCCAAU  981 2462-2482 AUUGGATUUUCGUAGCCGUUCUG  9822460-2482 AD-392846 AACGGCUACGAAAAUCCAACU  983 2463-2483AGUUGGAUUUUCGUAGCCGUUCU  984 2461-2483 AD-392859 ACGGCUACGAAAAUCCAACCU 985 2464-2484 AGGUUGGAUUUUCGUAGCCGUUC  986 2462-2484 AD-392843GGCUACGAAAAUCCAACCUAU  987 2466-2486 AUAGGUTGGAUUUUCGUAGCCGU  9882464-2486 AD-392855 GCUACGAAAAUCCAACCUACA  989 2467-2487UGUAGGTUGGAUUUUCGUAGCCG  990 2465-2487 AD-392840 CUACGAAAAUCCAACCUACAA 991 2468-2488 UUGUAGGUUGGAUUUUCGUAGCC  992 2466-2488 AD-392835UACGAAAAUCCAACCUACAAU  993 2469-2489 AUUGUAGGUUGGAUUUUCGUAGC  9942467-2489 AD-392729 ACGAAAAUCCAACCUACAAGU  995 2470-2490ACUUGUAGGUUGGAUUUUCGUAG  996 2468-2490 AD-392916 AAAAUCCAACCUACAAGUUCU 997 2473-2493 AGAACUTGUAGGUUGGAUUUUCG  998 2471-2493 AD-392876AAAUCCAACCUACAAGUUCUU  999 2474-2494 AAGAACTUGUAGGUUGGAUUUUC 10002472-2494 AD-392861 AUCCAACCUACAAGUUCUUUG 1001 2476-2496CAAAGAACUUGUAGGUUGGAUUU 1002 2474-2496 AD-392863 UCCAACCUACAAGUUCUUUGA1003 2477-2497 UCAAAGAACUUGUAGGUUGGAUU 1004 2475-2497 AD-392917CCAACCUACAAGUUCUUUGAU 1005 2478-2498 AUCAAAGAACUUGUAGGUUGGAU 10062476-2498 AD-392783 CCUCUGAAGUUGGACAGCAAA 1007 2530-2550UUUGCUGUCCAACUUCAGAGGCU 1008 2528-2550 AD-392765 AAGUUGGACAGCAAAACCAUU1009 2536-2556 AAUGGUTUUGCUGUCCAACUUCA 1010 2534-2556 AD-392791AGUUGGACAGCAAAACCAUUU 1011 2537-2557 AAAUGGTUUUGCUGUCCAACUUC 10122535-2557 AD-392800 UUGGACAGCAAAACCAUUGCU 1013 2539-2559AGCAAUGGUUUUGCUGUCCAACU 1014 2537-2559 AD-392711 GCAAAACCAUUGCUUCACUAU1015 2546-2566 AUAGUGAAGCAAUGGUUUUGCUG 1016 2544-2566 AD-392801AAACCAUUGCUUCACUACCCA 1017 2549-2569 UGGGUAGUGAAGCAAUGGUUUUG 10182547-2569 AD-392826 UACCCAUCGGUGUCCAUUUAU 1019 2564-2584AUAAAUGGACACCGAUGGGUAGU 1020 2562-2584 AD-392818 ACCCAUCGGUGUCCAUUUAUA1021 2565-2585 UAUAAATGGACACCGAUGGGUAG 1022 2563-2585 AD-392792CCCAUCGGUGUCCAUUUAUAU 1023 2566-2586 AUAUAAAUGGACACCGAUGGGUA 10242564-2586 AD-392802 CCAUCGGUGUCCAUUUAUAGA 1025 2567-2587UCUAUAAAUGGACACCGAUGGGU 1026 2565-2587 AD-392766 AUCGGUGUCCAUUUAUAGAAU1027 2569-2589 AUUCUATAAAUGGACACCGAUGG 1028 2567-2589 AD-392767UCGGUGUCCAUUUAUAGAAUA 1029 2570-2590 UAUUCUAUAAAUGGACACCGAUG 10302568-2590 AD-392834 ACCCGUUUUAUGAUUUACUCA 1031 2607-2627UGAGUAAAUCAUAAAACGGGUUU 1032 2605-2627 AD-392974 CCCGUUUUAUGAUUUACUCAU1033 2608-2628 AUGAGUAAAUCAUAAAACGGGUU 1034 2606-2628 AD-392784UUAUGAUUUACUCAUUAUCGU 1035 2614-2634 ACGAUAAUGAGUAAAUCAUAAAA 10362612-2634 AD-392744 AUGAUUUACUCAUUAUCGCCU 1037 2616-2636AGGCGATAAUGAGUAAAUCAUAA 1038 2614-2636 AD-392752 UGAUUUACUCAUUAUCGCCUU1039 2617-2637 AAGGCGAUAAUGAGUAAAUCAUA 1040 2615-2637 AD-392737GAUUUACUCAUUAUCGCCUUU 1041 2618-2638 AAAGGCGAUAAUGAGUAAAUCAU 10422616-2638 AD-392712 AUUUACUCAUUAUCGCCUUUU 1043 2619-2639AAAAGGCGAUAAUGAGUAAAUCA 1044 2617-2639 AD-392705 UUUACUCAUUAUCGCCUUUUG1045 2620-2640 CAAAAGGCGAUAAUGAGUAAAUC 1046 2618-2640 AD-392713UACUCAUUAUCGCCUUUUGAU 1047 2622-2642 AUCAAAAGGCGAUAAUGAGUAAA 10482620-2642 AD-392918 ACUCAUUAUCGCCUUUUGACA 1049 2623-2643UGUCAAAAGGCGAUAAUGAGUAA 1050 2621-2643 AD-392919 CUCAUUAUCGCCUUUUGACAU1051 2624-2644 AUGUCAAAAGGCGAUAAUGAGUA 1052 2622-2644 AD-392803UUAUCGCCUUUUGACAGCUGU 1053 2628-2648 ACAGCUGUCAAAAGGCGAUAAUG 10542626-2648 AD-392804 AUCGCCUUUUGACAGCUGUGU 1055 2630-2650ACACAGCUGUCAAAAGGCGAUAA 1056 2628-2650 AD-392827 UUUUGACAGCUGUGCUGUAAU1057 2636-2656 AUUACAGCACAGCUGUCAAAAGG 1058 2634-2656 AD-392828UUGACAGCUGUGCUGUAACAU 1059 2638-2658 AUGUUACAGCACAGCUGUCAAAA 10602636-2658 AD-392785 ACAGCUGUGCUGUAACACAAU 1061 2641-2661AUUGUGTUACAGCACAGCUGUCA 1062 2639-2661 AD-392829 AGCUGUGCUGUAACACAAGUA1063 2643-2663 UACUUGTGUUACAGCACAGCUGU 1064 2641-2663 AD-392920UGUGCUGUAACACAAGUAGAU 1065 2646-2666 AUCUACTUGUGUUACAGCACAGC 10662644-2666 AD-392921 GUGCUGUAACACAAGUAGAUU 1067 2647-2667AAUCUACUUGUGUUACAGCACAG 1068 2645-2667 AD-392768 GCUGUAACACAAGUAGAUGCU1069 2649-2669 AGCAUCTACUUGUGUUACAGCAC 1070 2647-2669 AD-392805ACACAAGUAGAUGCCUGAACU 1071 2655-2675 AGUUCAGGCAUCUACUUGUGUUA 10722653-2675 AD-392769 AAGUAGAUGCCUGAACUUGAA 1073 2659-2679UUCAAGTUCAGGCAUCUACUUGU 1074 2657-2679 AD-392753 GUAGAUGCCUGAACUUGAAUU1075 2661-2681 AAUUCAAGUUCAGGCAUCUACUU 1076 2659-2681 AD-392714UGCCUGAACUUGAAUUAAUCU 1077 2666-2686 AGAUUAAUUCAAGUUCAGGCAUC 10782664-2686 AD-392703 CCUGAACUUGAAUUAAUCCAU 1079 2668-2688AUGGAUTAAUUCAAGUUCAGGCA 1080 2666-2688 AD-392715 CUGAACUUGAAUUAAUCCACA1081 2669-2689 UGUGGATUAAUUCAAGUUCAGGC 1082 2667-2689 AD-392841AUCCACACAUCAGUAAUGUAU 1083 2683-2703 AUACAUTACUGAUGUGUGGAUUA 10842681-2703 AD-392836 UCCACACAUCAGUAAUGUAUU 1085 2684-2704AAUACATUACUGAUGUGUGGAUU 1086 2682-2704 AD-392966 CCACACAUCAGUAAUGUAUUU1087 2685-2705 AAAUACAUUACUGAUGUGUGGAU 1088 2683-2705 AD-392832CACACAUCAGUAAUGUAUUCU 1089 2686-2706 AGAAUACAUUACUGAUGUGUGGA 10902684-2706 AD-392972 ACACAUCAGUAAUGUAUUCUA 1091 2687-2707UAGAAUACAUUACUGAUGUGUGG 1092 2685-2707 AD-392961 UGUAUUCUAUCUCUCUUUACA1093 2699-2719 UGUAAAGAGAGAUAGAAUACAUU 1094 2697-2719 AD-392967CUAUCUCUCUUUACAUUUUGU 1095 2705-2725 ACAAAATGUAAAGAGAGAUAGAA 10962703-2725 AD-392893 UAUCUCUCUUUACAUUUUGGU 1097 2706-2726ACCAAAAUGUAAAGAGAGAUAGA 1098 2704-2726 AD-392894 AUCUCUCUUUACAUUUUGGUU1099 2707-2727 AACCAAAAUGUAAAGAGAGAUAG 1100 2705-2727 AD-392864UCUCUCUUUACAUUUUGGUCU 1101 2708-2728 AGACCAAAAUGUAAAGAGAGAUA 11022706-2728 AD-392865 CUCUCUUUACAUUUUGGUCUU 1103 2709-2729AAGACCAAAAUGUAAAGAGAGAU 1104 2707-2729 AD-392922 UCUUUACAUUUUGGUCUCUAU1105 2712-2732 AUAGAGACCAAAAUGUAAAGAGA 1106 2710-2732 AD-392833UGGUCUCUAUACUACAUUAUU 1107 2723-2743 AAUAAUGUAGUAUAGAGACCAAA 11082721-2743 AD-392968 GGUCUCUAUACUACAUUAUUA 1109 2724-2744UAAUAATGUAGUAUAGAGACCAA 1110 2722-2744 AD-392962 GUCUCUAUACUACAUUAUUAA1111 2725-2745 UUAAUAAUGUAGUAUAGAGACCA 1112 2723-2745 AD-392963UCUCUAUACUACAUUAUUAAU 1113 2726-2746 AUUAAUAAUGUAGUAUAGAGACC 11142724-2746 AD-392964 CUCUAUACUACAUUAUUAAUU 1115 2727-2747AAUUAATAAUGUAGUAUAGAGAC 1116 2725-2747 AD-392969 UCUAUACUACAUUAUUAAUGU1117 2728-2748 ACAUUAAUAAUGUAGUAUAGAGA 1118 2726-2748 AD-392973CUAUACUACAUUAUUAAUGGU 1119 2729-2749 ACCAUUAAUAAUGUAGUAUAGAG 11202727-2749 AD-392923 AUGGGUUUUGUGUACUGUAAA 1121 2745-2765UUUACAGUACACAAAACCCAUUA 1122 2743-2765 AD-392866 UUUGUGUACUGUAAAGAAUUU1123 2751-2771 AAAUUCTUUACAGUACACAAAAC 1124 2749-2771 AD-392924UUGUGUACUGUAAAGAAUUUA 1125 2752-2772 UAAAUUCUUUACAGUACACAAAA 11262750-2772 AD-392895 UGUGUACUGUAAAGAAUUUAU 1127 2753-2773AUAAAUTCUUUACAGUACACAAA 1128 2751-2773 AD-392867 GUGUACUGUAAAGAAUUUAGU1129 2754-2774 ACUAAATUCUUUACAGUACACAA 1130 2752-2774 AD-392877GUACUGUAAAGAAUUUAGCUU 1131 2756-2776 AAGCUAAAUUCUUUACAGUACAC 11322754-2776 AD-392707 AUUUAGCUGUAUCAAACUAGU 1133 2768-2788ACUAGUTUGAUACAGCUAAAUUC 1134 2766-2788 AD-392716 UUUAGCUGUAUCAAACUAGUU1135 2769-2789 AACUAGTUUGAUACAGCUAAAUU 1136 2767-2789 AD-392925GCUGUAUCAAACUAGUGCAUU 1137 2773-2793 AAUGCACUAGUUUGAUACAGCUA 11382771-2793 AD-392926 CUAGUGCAUGAAUAGAUUCUU 1139 2784-2804AAGAAUCUAUUCAUGCACUAGUU 1140 2782-2804 AD-392927 UAGUGCAUGAAUAGAUUCUCU1141 2785-2805 AGAGAATCUAUUCAUGCACUAGU 1142 2783-2805 AD-392717GAAUAGAUUCUCUCCUGAUUA 1143 2793-2813 UAAUCAGGAGAGAAUCUAUUCAU 11442791-2813 AD-392928 CUCUCCUGAUUAUUUAUCACA 1145 2802-2822UGUGAUAAAUAAUCAGGAGAGAA 1146 2800-2822 AD-392700 UCUCCUGAUUAUUUAUCACAU1147 2803-2823 AUGUGATAAAUAAUCAGGAGAGA 1148 2801-2823 AD-392878CUCCUGAUUAUUUAUCACAUA 1149 2804-2824 UAUGUGAUAAAUAAUCAGGAGAG 11502802-2824 AD-392718 UCCUGAUUAUUUAUCACAUAU 1151 2805-2825AUAUGUGAUAAAUAAUCAGGAGA 1152 2803-2825 AD-392929 CCUGAUUAUUUAUCACAUAGU1153 2806-2826 ACUAUGTGAUAAAUAAUCAGGAG 1154 2804-2826 AD-392879GCCAGUUGUAUAUUAUUCUUU 1155 2833-2853 AAAGAATAAUAUACAACUGGCUA 11562831-2853 AD-392754 UUGUAUAUUAUUCUUGUGGUU 1157 2838-2858AACCACAAGAAUAAUAUACAACU 1158 2836-2858 AD-392819 UCUUGUGGUUUGUGACCCAAU1159 2849-2869 AUUGGGTCACAAACCACAAGAAU 1160 2847-2869 AD-392745CUUGUGGUUUGUGACCCAAUU 1161 2850-2870 AAUUGGGUCACAAACCACAAGAA 11622848-2870 AD-392770 UUGUGGUUUGUGACCCAAUUA 1163 2851-2871UAAUUGGGUCACAAACCACAAGA 1164 2849-2871 AD-392806 UGUGGUUUGUGACCCAAUUAA1165 2852-2872 UUAAUUGGGUCACAAACCACAAG 1166 2850-2872 AD-392771GUUUGUGACCCAAUUAAGUCU 1167 2856-2876 AGACUUAAUUGGGUCACAAACCA 11682854-2876 AD-392820 UUUGUGACCCAAUUAAGUCCU 1169 2857-2877AGGACUTAAUUGGGUCACAAACC 1170 2855-2877 AD-392821 UUGUGACCCAAUUAAGUCCUA1171 2858-2878 UAGGACTUAAUUGGGUCACAAAC 1172 2856-2878 AD-392786UGUGACCCAAUUAAGUCCUAU 1173 2859-2879 AUAGGACUUAAUUGGGUCACAAA 11742857-2879 AD-392772 GUGACCCAAUUAAGUCCUACU 1175 2860-2880AGUAGGACUUAAUUGGGUCACAA 1176 2858-2880 AD-392699 GACCCAAUUAAGUCCUACUUU1177 2862-2882 AAAGUAGGACUUAAUUGGGUCAC 1178 2860-2882 AD-392868ACCCAAUUAAGUCCUACUUUA 1179 2863-2883 UAAAGUAGGACUUAAUUGGGUCA 11802861-2883 AD-392719 CCCAAUUAAGUCCUACUUUAU 1181 2864-2884AUAAAGTAGGACUUAAUUGGGUC 1182 2862-2884 AD-392880 AAUUAAGUCCUACUUUACAUA1183 2867-2887 UAUGUAAAGUAGGACUUAAUUGG 1184 2865-2887 AD-392930UAAGUCCUACUUUACAUAUGU 1185 2870-2890 ACAUAUGUAAAGUAGGACUUAAU 11862868-2890 AD-392931 AGUCCUACUUUACAUAUGCUU 1187 2872-2892AAGCAUAUGUAAAGUAGGACUUA 1188 2870-2892 AD-392932 GUCCUACUUUACAUAUGCUUU1189 2873-2893 AAAGCATAUGUAAAGUAGGACUU 1190 2871-2893 AD-392869UCCUACUUUACAUAUGCUUUA 1191 2874-2894 UAAAGCAUAUGUAAAGUAGGACU 11922872-2894 AD-392870 CCUACUUUACAUAUGCUUUAA 1193 2875-2895UUAAAGCAUAUGUAAAGUAGGAC 1194 2873-2895 AD-392896 CUACUUUACAUAUGCUUUAAU1195 2876-2896 AUUAAAGCAUAUGUAAAGUAGGA 1196 2874-2896 AD-392787UACAUAUGCUUUAAGAAUCGA 1197 2882-2902 UCGAUUCUUAAAGCAUAUGUAAA 11982880-2902 AD-392720 CAUAUGCUUUAAGAAUCGAUU 1199 2884-2904AAUCGATUCUUAAAGCAUAUGUA 1200 2882-2904 AD-392746 AUAUGCUUUAAGAAUCGAUGU1201 2885-2905 ACAUCGAUUCUUAAAGCAUAUGU 1202 2883-2905 AD-392773UAUGCUUUAAGAAUCGAUGGU 1203 2886-2906 ACCAUCGAUUCUUAAAGCAUAUG 12042884-2906 AD-392807 GGGAUGCUUCAUGUGAACGUU 1205 2906-2926AACGUUCACAUGAAGCAUCCCCC 1206 2904-2926 AD-392730 UGCUUCUCUUGCCUAAGUAUU1207 2937-2957 AAUACUTAGGCAAGAGAAGCAGC 1208 2935-2957 AD-392721CUUCUCUUGCCUAAGUAUUCU 1209 2939-2959 AGAAUACUUAGGCAAGAGAAGCA 12102937-2959 AD-392933 UUCUCUUGCCUAAGUAUUCCU 1211 2940-2960AGGAAUACUUAGGCAAGAGAAGC 1212 2938-2960 AD-392934 CUCUUGCCUAAGUAUUCCUUU1213 2942-2962 AAAGGAAUACUUAGGCAAGAGAA 1214 2940-2962 AD-392881CUUGCCUAAGUAUUCCUUUCU 1215 2944-2964 AGAAAGGAAUACUUAGGCAAGAG 12162942-2964 AD-392897 UGCCUAAGUAUUCCUUUCCUU 1217 2946-2966AAGGAAAGGAAUACUUAGGCAAG 1218 2944-2966 AD-392898 AAGUAUUCCUUUCCUGAUCAU1219 2951-2971 AUGAUCAGGAAAGGAAUACUUAG 1220 2949-2971 AD-392708AGUAUUCCUUUCCUGAUCACU 1221 2952-2972 AGUGAUCAGGAAAGGAAUACUUA 12222950-2972 AD-392899 GUAUUCCUUUCCUGAUCACUA 1223 2953-2973UAGUGATCAGGAAAGGAAUACUU 1224 2951-2973 AD-392935 UAUUCCUUUCCUGAUCACUAU1225 2954-2974 AUAGUGAUCAGGAAAGGAAUACU 1226 2952-2974 AD-392882AUUCCUUUCCUGAUCACUAUU 1227 2955-2975 AAUAGUGAUCAGGAAAGGAAUAC 12282953-2975 AD-392738 UCCUUUCCUGAUCACUAUGCA 1229 2957-2977UGCAUAGUGAUCAGGAAAGGAAU 1230 2955-2977 AD-392739 CUUUCCUGAUCACUAUGCAUU1231 2959-2979 AAUGCATAGUGAUCAGGAAAGGA 1232 2957-2979 AD-392936UUUCCUGAUCACUAUGCAUUU 1233 2960-2980 AAAUGCAUAGUGAUCAGGAAAGG 12342958-2980 AD-392900 UUCCUGAUCACUAUGCAUUUU 1235 2961-2981AAAAUGCAUAGUGAUCAGGAAAG 1236 2959-2981 AD-392901 CUGAUCACUAUGCAUUUUAAA1237 2964-2984 UUUAAAAUGCAUAGUGAUCAGGA 1238 2962-2984 AD-392937CACUAUGCAUUUUAAAGUUAA 1239 2969-2989 UUAACUTUAAAAUGCAUAGUGAU 12402967-2989 AD-392883 ACUAUGCAUUUUAAAGUUAAA 1241 2970-2990UUUAACTUUAAAAUGCAUAGUGA 1242 2968-2990 AD-392975 UUCCAUGACUGCAUUUUACUU1243 3029-3049 AAGUAAAAUGCAGUCAUGGAAAA 1244 3027-3049 AD-392938CUGCAUUUUACUGUACAGAUU 1245 3037-3057 AAUCUGTACAGUAAAAUGCAGUC 12463035-3057 AD-392755 AUUGCUGCUUCUGCUAUAUUU 1247 3055-3075AAAUAUAGCAGAAGCAGCAAUCU 1248 3053-3075 AD-392939 UUCUGCUAUAUUUGUGAUAUA1249 3063-3083 UAUAUCACAAAUAUAGCAGAAGC 1250 3061-3083 AD-392940UCUGCUAUAUUUGUGAUAUAU 1251 3064-3084 AUAUAUCACAAAUAUAGCAGAAG 12523062-3084 AD-392756 UGCUAUAUUUGUGAUAUAGGA 1253 3066-3086UCCUAUAUCACAAAUAUAGCAGA 1254 3064-3086 AD-392774 UUUGUGAUAUAGGAAUUAAGA1255 3073-3093 UCUUAATUCCUAUAUCACAAAUA 1256 3071-3093 AD-392850UCUUCGUGCCUGUUUUAUGUU 1257 3111-3131 AACAUAAAACAGGCACGAAGAAA 12583109-3131 AD-392852 CUUCGUGCCUGUUUUAUGUGU 1259 3112-3132ACACAUAAAACAGGCACGAAGAA 1260 3110-3132 AD-392830 GUUUUAUGUGCACACAUUAGU1261 3122-3142 ACUAAUGUGUGCACAUAAAACAG 1262 3120-3142 AD-392808UGUGCACACAUUAGGCAUUGA 1263 3128-3148 UCAAUGCCUAAUGUGUGCACAUA 12643126-3148 AD-392793 UGCACACAUUAGGCAUUGAGA 1265 3130-3150UCUCAATGCCUAAUGUGUGCACA 1266 3128-3150 AD-392757 ACACAUUAGGCAUUGAGACUU1267 3133-3153 AAGUCUCAAUGCCUAAUGUGUGC 1268 3131-3153 AD-392747UUUGUCCACGUAUCUUUGGGU 1269 3168-3188 ACCCAAAGAUACGUGGACAAAAA 12703166-3188 AD-392902 CACGUAUCUUUGGGUCUUUGA 1271 3174-3194UCAAAGACCCAAAGAUACGUGGA 1272 3172-3194 AD-392941 ACGUAUCUUUGGGUCUUUGAU1273 3175-3195 AUCAAAGACCCAAAGAUACGUGG 1274 3173-3195 AD-392942UCUUUGGGUCUUUGAUAAAGA 1275 3180-3200 UCUUUATCAAAGACCCAAAGAUA 12763178-3200 AD-392943 CUUUGGGUCUUUGAUAAAGAA 1277 3181-3201UUCUUUAUCAAAGACCCAAAGAU 1278 3179-3201 AD-392944 UUGGGUCUUUGAUAAAGAAAA1279 3183-3203 UUUUCUTUAUCAAAGACCCAAAG 1280 3181-3203 AD-392903UGGGUCUUUGAUAAAGAAAAU 1281 3184-3204 AUUUUCTUUAUCAAAGACCCAAA 12823182-3204 AD-392775 AAAGAAUCCCUGUUCAUUGUA 1283 3201-3221UACAAUGAACAGGGAUUCUUUUC 1284 3199-3221 AD-392758 AAGAAUCCCUGUUCAUUGUAA1285 3202-3222 UUACAATGAACAGGGAUUCUUUU 1286 3200-3222 AD-392945AGAAUCCCUGUUCAUUGUAAU 1287 3203-3223 AUUACAAUGAACAGGGAUUCUUU 12883201-3223 AD-392946 GAAUCCCUGUUCAUUGUAAGU 1289 3204-3224ACUUACAAUGAACAGGGAUUCUU 1290 3202-3224 AD-392884 UGUUCAUUGUAAGCACUUUUA1291 3211-3231 UAAAAGTGCUUACAAUGAACAGG 1292 3209-3231 AD-392947GUUCAUUGUAAGCACUUUUAU 1293 3212-3232 AUAAAAGUGCUUACAAUGAACAG 12943210-3232 AD-392748 UCAUUGUAAGCACUUUUACGU 1295 3214-3234ACGUAAAAGUGCUUACAAUGAAC 1296 3212-3234 AD-392759 CAUUGUAAGCACUUUUACGGU1297 3215-3235 ACCGUAAAAGUGCUUACAAUGAA 1298 3213-3235 AD-392837CUGGUCUUCAAUUACCAAGAA 1299 3258-3278 UUCUUGGUAAUUGAAGACCAGCA 13003256-3278 AD-392970 GGUCUUCAAUUACCAAGAAUU 1301 3260-3280AAUUCUTGGUAAUUGAAGACCAG 1302 3258-3280 AD-392976 UCUUCAAUUACCAAGAAUUCU1303 3262-3282 AGAAUUCUUGGUAAUUGAAGACC 1304 3260-3282 AD-392965CUUCAAUUACCAAGAAUUCUU 1305 3263-3283 AAGAAUTCUUGGUAAUUGAAGAC 13063261-3283 AD-392831 UUCAAUUACCAAGAAUUCUCU 1307 3264-3284AGAGAATUCUUGGUAAUUGAAGA 1308 3262-3284 AD-392904 UCAAUUACCAAGAAUUCUCCA1309 3265-3285 UGGAGAAUUCUUGGUAAUUGAAG 1310 3263-3285 AD-392885AAUUACCAAGAAUUCUCCAAA 1311 3267-3287 UUUGGAGAAUUCUUGGUAAUUGA 13123265-3287 AD-392886 UUACCAAGAAUUCUCCAAAAU 1313 3269-3289AUUUUGGAGAAUUCUUGGUAAUU 1314 3267-3289 AD-392776 UGAUUGUACAGAAUCAUUGCU1315 3304-3324 AGCAAUGAUUCUGUACAAUCAUC 1316 3302-3324 AD-392887UCAUUGCUUAUGACAUGAUCU 1317 3317-3337 AGAUCATGUCAUAAGCAAUGAUU 13183315-3337 AD-392722 CAUUGCUUAUGACAUGAUCGU 1319 3318-3338ACGAUCAUGUCAUAAGCAAUGAU 1320 3316-3338 AD-392740 AUUGCUUAUGACAUGAUCGCU1321 3319-3339 AGCGAUCAUGUCAUAAGCAAUGA 1322 3317-3339 AD-392760UUGCUUAUGACAUGAUCGCUU 1323 3320-3340 AAGCGATCAUGUCAUAAGCAAUG 13243318-3340 AD-392731 UGCUUAUGACAUGAUCGCUUU 1325 3321-3341AAAGCGAUCAUGUCAUAAGCAAU 1326 3319-3341 AD-392709 GCUUAUGACAUGAUCGCUUUC1327 3322-3342 GAAAGCGAUCAUGUCAUAAGCAA 1328 3320-3342 AD-392723CUUAUGACAUGAUCGCUUUCU 1329 3323-3343 AGAAAGCGAUCAUGUCAUAAGCA 13303321-3343 AD-392948 UUAUGACAUGAUCGCUUUCUA 1331 3324-3344UAGAAAGCGAUCAUGUCAUAAGC 1332 3322-3344 AD-392724 UAUGACAUGAUCGCUUUCUAU1333 3325-3345 AUAGAAAGCGAUCAUGUCAUAAG 1334 3323-3345 AD-392949AUGACAUGAUCGCUUUCUACA 1335 3326-3346 UGUAGAAAGCGAUCAUGUCAUAA 13363324-3346 AD-392725 UGACAUGAUCGCUUUCUACAU 1337 3327-3347AUGUAGAAAGCGAUCAUGUCAUA 1338 3325-3347 AD-392950 CAUGAUCGCUUUCUACACUGU1339 3330-3350 ACAGUGTAGAAAGCGAUCAUGUC 1340 3328-3350 AD-392732UGAUCGCUUUCUACACUGUAU 1341 3332-3352 AUACAGTGUAGAAAGCGAUCAUG 13423330-3352 AD-392726 GAUCGCUUUCUACACUGUAUU 1343 3333-3353AAUACAGUGUAGAAAGCGAUCAU 1344 3331-3353 AD-392733 AUCGCUUUCUACACUGUAUUA1345 3334-3354 UAAUACAGUGUAGAAAGCGAUCA 1346 3332-3354 AD-392906UCGCUUUCUACACUGUAUUAU 1347 3335-3355 AUAAUACAGUGUAGAAAGCGAUC 13483333-3355 AD-392862 CGCUUUCUACACUGUAUUACA 1349 3336-3356UGUAAUACAGUGUAGAAAGCGAU 1350 3334-3356 AD-392951 CUUUCUACACUGUAUUACAUA1351 3338-3358 UAUGUAAUACAGUGUAGAAAGCG 1352 3336-3358 AD-392871UUCUACACUGUAUUACAUAAA 1353 3340-3360 UUUAUGTAAUACAGUGUAGAAAG 13543338-3360 AD-392872 UCUACACUGUAUUACAUAAAU 1355 3341-3361AUUUAUGUAAUACAGUGUAGAAA 1356 3339-3361 AD-392952 GAUUCAAUUUUCUUUAACCAU1357 3456-3476 AUGGUUAAAGAAAAUUGAAUCUG 1358 3454-3476 AD-392907AUUUUCUUUAACCAGUCUGAA 1359 3462-3482 UUCAGACUGGUUAAAGAAAAUUG 13603460-3482 AD-392953 UUUCUUUAACCAGUCUGAAGU 1361 3464-3484ACUUCAGACUGGUUAAAGAAAAU 1362 3462-3484 AD-392741 UCUUUAACCAGUCUGAAGUUU1363 3466-3486 AAACUUCAGACUGGUUAAAGAAA 1364 3464-3486 AD-392908CUUUAACCAGUCUGAAGUUUC 1365 3467-3487 GAAACUTCAGACUGGUUAAAGAA 13663465-3487 AD-392977 CUGAAGUUUCAUUUAUGAUAU 1367 3478-3498AUAUCATAAAUGAAACUUCAGAC 1368 3476-3498 AD-392847 GAAGUUUCAUUUAUGAUACAA1369 3480-3500 UUGUAUCAUAAAUGAAACUUCAG 1370 3478-3500 AD-392809AAAUGGAAGUGGCAAUAUAAU 1371 3511-3531 AUUAUATUGCCACUUCCAUUUUC 13723509-3531 AD-392810 AUGGAAGUGGCAAUAUAAGGU 1373 3513-3533ACCUUATAUUGCCACUUCCAUUU 1374 3511-3533 AD-392777 UGCCUGGACAAACCCUUCUUU1375 3547-3567 AAAGAAGGGUUUGUCCAGGCAUG 1376 3545-3567 AD-392960UUCUUUUAAGAUGUGUCUUCA 1377 3562-3582 UGAAGACACAUCUUAAAAGAAGG 13783560-3582 AD-392873 CUUUUAAGAUGUGUCUUCAAU 1379 3564-3584AUUGAAGACACAUCUUAAAAGAA 1380 3562-3584 AD-392889 UUUUAAGAUGUGUCUUCAAUU1381 3565-3585 AAUUGAAGACACAUCUUAAAAGA 1382 3563-3585 AD-392954UUUAAGAUGUGUCUUCAAUUU 1383 3566-3586 AAAUUGAAGACACAUCUUAAAAG 13843564-3586 AD-392955 UUAAGAUGUGUCUUCAAUUUG 1385 3567-3587CAAAUUGAAGACACAUCUUAAAA 1386 3565-3587 AD-392909 UAAGAUGUGUCUUCAAUUUGU1387 3568-3588 ACAAAUTGAAGACACAUCUUAAA 1388 3566-3588 AD-392710AAGAUGUGUCUUCAAUUUGUA 1389 3569-3589 UACAAATUGAAGACACAUCUUAA 13903567-3589 AD-392956 AGAUGUGUCUUCAAUUUGUAU 1391 3570-3590AUACAAAUUGAAGACACAUCUUA 1392 3568-3590 AD-392874 AUGUGUCUUCAAUUUGUAUAA1393 3572-3592 UUAUACAAAUUGAAGACACAUCU 1394 3570-3592 AD-392957UGUCUUCAAUUUGUAUAAAAU 1395 3575-3595 AUUUUATACAAAUUGAAGACACA 13963573-3595 AD-392958 CUUCAAUUUGUAUAAAAUGGU 1397 3578-3598ACCAUUTUAUACAAAUUGAAGAC 1398 3576-3598 AD-392959 AUGGUGUUUUCAUGUAAAUAA1399 3594-3614 UUAUUUACAUGAAAACACCAUUU 1400 3592-3614 AD-392788GUAAAUAAAUACAUUCUUGGA 1401 3607-3627 UCCAAGAAUGUAUUUAUUUACAU 14023605-3627

TABLE 4 APP Single Dose Screen in Primary Cynomolgus Hepatocytes andBe(2)C Cell Line Data are expressed as percent message remainingrelative to AD-1955 non-targeting control. Primary CynomolgusHepatocytes Be(2)C Cell Line Duplex 10 nM 10 nM 0.1 nM 0.1 nM 10 nM 10nM 0.1 nM 0.1 nM Name Avg SD Avg SD Avg SD Avg SD AD-392853 92 5 89.91.5 97 2.5 99.3 8.8 AD-392857 86.7 3.3 98.9 6.1 85.1 4.4 103.8 5.9AD-392851 90.5 1.5 97.9 10.1 100.1 4 103.9 7.8 AD-392811 90.5 10.5 87.82.5 89.1 6.8 98 5.1 AD-392910 52.3 3 99.2 32.4 66.1 6.1 101.3 9.7AD-392890 57.4 4.8 108.5 23.1 63.9 1.5 100.3 10.6 AD-392911 16.4 3.485.7 4 10.6 3.5 71.2 10.3 AD-392912 16.7 2.7 84.8 4.5 9.7 1.7 57.7 4.1AD-392778 46.1 19.2 96 23.4 7.9 0.9 82.4 7.4 AD-392727 52.9 5.8 98.911.4 48.3 4.5 94 5.7 AD-392728 43.8 20.3 91.5 10.2 17.6 2.2 86.2 6.5AD-392891 52 7 142.2 35.1 34.8 1.7 93.5 5.8 AD-392822 53.9 3.8 75.2 2.930.1 3.2 83.7 5.8 AD-392749 46.3 11.7 97.6 2.6 14.9 1.7 95.7 5.3AD-392794 108.8 17.9 86.9 2.7 92.9 7.9 87.4 6.7 AD-392795 39.5 13.2 78.111.8 15.5 1.8 79.9 7.9 AD-392812 87.2 4.3 90.4 2.5 79.8 3.3 78.5 13.8AD-392796 48 17.6 82.6 2.8 17.1 2.5 80.2 3.5 AD-392779 100 30.9 95.9 4.899.6 4 98.6 3.3 AD-392780 80.7 29.5 93.2 4.5 47.4 4.4 101.6 5.2AD-392813 91.6 2.9 85.1 4 84.8 4.7 88.9 7 AD-392797 98 6.6 88.7 11.1 793.3 84 12 AD-392761 73.9 18.4 94.2 4.3 77.9 4.4 101 6.4 AD-392814 56.92.9 84.4 5.4 47.5 2.6 83.8 6.6 AD-392742 89 21.9 99.4 8.2 48.1 5.8 96.63.7 AD-392750 110.7 44.7 99.9 13.2 25.4 1.2 95 4.7 AD-392823 65.5 3 73.72.9 38.8 4.1 84.9 3.8 AD-392789 103.7 4 105 3.8 88.1 7 79.5 4 AD-39278181 39.1 94.9 5.8 21.2 3.1 95 8.9 AD-392798 119.2 16.3 85.3 10.9 73.1 6.383.2 7.4 AD-392751 48.5 12.9 93.9 7.9 15.6 3 87.2 2.5 AD-392858 90 1.595 2.6 90.7 4.7 103 7.7 AD-392844 21.8 0.4 93 3.6 6.2 0.6 51.8 5.3AD-392842 88.9 0.5 98.2 1.6 67.7 4.1 102 2.7 AD-392848 91.7 9.1 90.1 2.670.9 7.5 96.5 16.7 AD-392838 68 3.6 90.2 3.3 20.2 2 84.3 6.2 AD-39283969 2.6 84.8 3.9 62.7 3.1 85.8 7.6 AD-392734 103 32.4 112.8 23.5 86.6 6.698.6 3.1 AD-392790 34 4.8 99.2 1.2 10.9 1.4 72.6 2.5 AD-392815 37.4 1.782.5 2.9 21.5 1.9 79.8 0.9 AD-392762 72.2 21.3 95 12.3 91.2 4.6 102.67.7 AD-392735 47 9.7 101.5 9.2 29.6 4.4 94 7.4 AD-392743 73.6 23.4 105.516.6 58.5 2.6 100.1 11.3 AD-392736 50.5 9 97.3 8.2 19.6 2.4 91.7 7AD-392824 22.6 6.7 65.8 4.9 6.4 1.6 54.9 5 AD-392799 90.1 23.6 75.8 4.535.7 5.4 78.2 7.5 AD-392971 89.2 13.4 92.1 0.3 57.1 3.6 91.8 5.8AD-392913 18.4 2.7 78.1 8 7.4 0.2 45.7 2.1 AD-392892 61 12.4 113.2 8.657.4 5.4 89.7 13.2 AD-392914 80.3 6.3 103.2 5.9 86.5 3.4 111.4 19.7AD-392860 91.8 4.8 89.4 6.1 106.1 6.2 98.6 5.6 AD-392875 96.2 4.8 107.92.5 66.1 2.9 83.5 8.4 AD-392915 48.1 1.8 101.9 4.8 38.3 3.4 103 5.4AD-392782 109.4 4.8 95.4 5.3 72.2 4.3 101.6 2.7 AD-392763 60 17.6 93 6.326.7 2.2 91.6 3.8 AD-392816 40.2 1.5 74.6 2.2 15.6 1.2 78.9 2.4AD-392704 28.7 12.1 94.1 6.8 15.8 1.5 65.7 9.7 AD-392854 89 3.5 84.9 2.999 7 97.9 5.8 AD-392856 93.7 2.5 88.4 2.8 101 7.8 94.2 3.5 AD-392817101.6 3 85 5.2 77.5 11.4 98.6 11.6 AD-392764 69.5 12.1 87.2 5.9 10.6 1.479.4 5.7 AD-392845 89.5 2 99 8.2 50.4 5 90.5 2.9 AD-392825 38.1 2.5 988.4 14.7 4.7 91.4 4 AD-392849 89.4 4.1 92.3 11.4 30.3 2.3 103.4 7.4AD-392846 83.1 1.9 99.7 6.3 17.6 3.2 77.7 4.2 AD-392859 82 2.5 91.4 5.569.7 1.5 98.6 2.1 AD-392843 18.8 2.1 88.9 5.4 7.4 2.5 37.2 2.2 AD-39285564 5.2 85.9 12.4 23.4 2.6 85.6 9.1 AD-392840 74.3 2.3 91.2 6.4 27.7 2.594.3 15.6 AD-392835 18.2 2.3 84.3 5.4 12.7 3.1 53.5 4.5 AD-392729 46.913.7 100.9 20.5 13.3 2.3 82.4 4.2 AD-392916 20 1.6 63.7 3.6 7.5 2 44.42.1 AD-392876 45.8 4.6 100.8 2.6 16.4 3.6 67.4 7.2 AD-392861 91.9 3.989.3 2.6 89.9 10.9 91.5 4.3 AD-392863 22.8 0.6 90.1 9.3 9.9 1.9 72.2 8AD-392917 30.6 1.8 99.7 2.1 21.7 3.5 82.5 7.5 AD-392783 22.8 1.7 90.411.1 13.1 1.4 69.8 5.7 AD-392765 79 22 83.3 6.4 22.4 2.8 68.1 5.7AD-392791 31.9 7.6 84.1 4.8 11.2 1.2 52.3 2.4 AD-392800 38.2 3.6 72.37.6 8 1.5 65.4 7.2 AD-392711 38.1 24.1 115.1 21 18.8 0.6 67.2 2.2AD-392801 18.7 0.6 87 6.3 11.7 3 66.3 17.5 AD-392826 69 4.6 95.1 10 31.93.3 88.4 8 AD-392818 31.5 2.2 77.8 6.6 18.6 3 80.7 6.2 AD-392792 35.86.7 87.7 4.1 10.7 1.1 58.3 4.7 AD-392802 43.8 4.1 81.8 7.5 26.5 3.7 90.32.6 AD-392766 32.8 11.5 75.2 4.1 8.4 2 38.1 3.5 AD-392767 64 23.5 87.55.2 10.7 1.5 66.1 5.8 AD-392834 84.6 2.8 85.1 6.9 7.8 0.8 68.1 4.7AD-392974 118.3 5.4 105.4 6.3 9.3 0.9 53.1 4.5 AD-392784 63.6 14.9 92.80.8 28.1 3.4 96.7 6.5 AD-392744 59.6 17.2 96.6 7.4 18.3 1 92.7 7.7AD-392752 38.2 11.6 92.8 4.9 7.7 1.2 57.6 2.3 AD-392737 44.8 38.6 103.927.2 9.7 0.7 57.3 3.4 AD-392712 73 38.4 102.8 6.1 37.2 1.9 67.4 16AD-392705 25.2 9.4 88.7 4.3 6.6 0.9 47.7 6.3 AD-392713 81.8 33.4 101.17.3 61.7 5.8 92.7 9.8 AD-392918 25.1 1.8 93.5 5.3 18.5 1 95 11.2AD-392919 24.3 3.3 95 8.6 13.8 4 78 9.1 AD-392803 51.5 3.1 89.5 9.4 19.82 72 3 AD-392804 72 3.3 97.2 11.3 22.9 1.2 83.1 3.2 AD-392827 24.1 1.587 9.2 11.7 1.7 72.7 5.9 AD-392828 67.5 3.7 102.4 13.8 33.7 3.2 81.9 3.9AD-392785 39.5 14.4 70.2 15 5.6 1.2 37.4 3.9 AD-392829 26.5 2.8 87.5 7.516.1 1.6 73 7.4 AD-392920 35.8 3.5 108.1 4.7 19.9 4.3 94.4 6.7 AD-39292130 3.8 100.7 9.1 11.9 2.8 75 7.6 AD-392768 66.5 21.9 94.1 6.6 13.1 2.784.9 5.8 AD-392805 20.5 0.9 88.7 13.4 7.9 2.2 43.5 3.9 AD-392769 41.921.5 74.6 4.6 4.9 2.1 32.5 3.9 AD-392753 40.4 7.6 113.9 21.9 12.5 0.972.5 7.6 AD-392714 21.7 8.1 99.5 7.2 6.9 0.8 40.8 3.2 AD-392703 17.6 1.590.5 6.9 6.2 1.4 37.7 3.9 AD-392715 25.5 10.3 78.8 4.8 6.4 1.7 38.9 2.7AD-392841 89.6 3.9 93.6 9 36.8 4.1 96.6 6.9 AD-392836 88.5 1.6 97.7 8.67.6 2 51.5 2 AD-392966 71.5 4.6 92.4 3.4 6.4 1 47.6 4.2 AD-392832 94.77.9 85.4 14.4 23.8 3.2 76.2 2.6 AD-392972 84.1 10.8 89.8 7.1 8.3 2 57.13.5 AD-392961 82.6 7.5 111.3 9.9 8 0.4 51.7 5.1 AD-392967 81.6 7.1 93.26.8 20.2 1.6 89.4 4.9 AD-392893 64.8 11.7 118.8 19.7 59.9 2.6 80.7 5.1AD-392894 68.4 10.3 111.4 10.8 21.9 1.5 88.4 15.6 AD-392864 62.7 15.488.4 6.2 8.2 0.8 55.9 4.5 AD-392865 45.8 2.4 103.8 12.6 13.6 3.1 35.85.4 AD-392922 43.3 5 106.5 2.2 11.1 5.2 53.1 4.9 AD-392833 95.1 5.1 93.94.1 21.2 0.7 86.2 0.8 AD-392968 54.3 3.1 94.8 9.3 8.2 0.7 51.9 2.5AD-392962 82.3 10.9 103 10 8.5 0.5 55 3.8 AD-392963 63.9 8.9 99.6 10.319.5 0.5 71.2 1.1 AD-392964 94.4 8.6 97.5 9.2 52.4 3.7 87.1 2.8AD-392969 73.3 6.6 99 6.2 11.7 1.1 69.4 2.5 AD-392973 69 12.8 87.7 8 7.60.7 67.3 1.7 AD-392923 28.6 3.3 106 8.2 13.2 3.5 69.6 12.7 AD-392866 184.3 86.5 14.1 9.1 0.8 29.1 8.6 AD-392924 79.7 3.1 108.3 5.2 89 3.1 94.87.7 AD-392895 63.4 13.8 109 4.4 31.6 2.9 86.7 8.9 AD-392867 95.2 11.699.8 15.8 45.3 1.7 77.1 6.6 AD-392877 74.8 23.6 102.2 7.6 14.3 2 54.11.7 AD-392707 27.1 7.6 87.9 5.5 6 1.4 68.8 1.9 AD-392716 107.6 19.9100.9 7.9 45.4 4 94.6 3.6 AD-392925 47 5.6 106.8 5.1 23.1 2.4 80.7 9.3AD-392926 22.1 2.5 93.7 8.7 7.7 0.7 67 9.8 AD-392927 18.2 5.4 80.1 8.79.7 2 44.2 6.4 AD-392717 57.4 16 84.6 9.4 8.7 0.9 52.2 3.7 AD-39292871.3 4 95.4 4.1 35.3 2.7 103 8.5 AD-392700 23 7.6 88.4 4.8 6.3 0.6 45.310.7 AD-392878 29.9 18.4 89 4.4 8.4 1.6 34.5 4 AD-392718 40.3 14.5 105.425.7 10.8 0.6 68.5 2.3 AD-392929 42.4 3.7 99.5 1.2 15 4.9 88.8 14.1AD-392879 102.2 14.5 97.7 3.5 59.6 3 67.3 8.1 AD-392754 97.1 14.7 102.117.6 27.3 2.5 108.6 5.7 AD-392819 22.3 2.2 79.6 4.9 11 2.5 58.4 4.9AD-392745 13.8 2.2 74 13.1 7.1 1.9 28.2 4 AD-392770 36.9 18 80.3 8.1 6.71 34.1 4.1 AD-392806 44.9 3.3 84.2 3.9 17.7 2.6 54.3 1.9 AD-392771 49.418.6 89.4 1.6 9.5 0.4 60.1 2.9 AD-392820 54.4 3.3 88.1 3.9 19.6 1.1 78.16.4 AD-392821 61.1 2.2 79.8 3.1 15.5 1.6 80.1 5.3 AD-392786 72.2 9.8109.4 4 19.8 1.9 65.3 2.2 AD-392772 58.9 11.7 88.9 2.6 11 0.6 62.2 3.1AD-392699 37.9 9.1 102.9 8.7 8.1 3.4 55.6 4.4 AD-392868 52.9 1.4 95.811.1 18 1.8 61.5 4.3 AD-392719 37.4 20.3 94.7 12.4 7.3 1 38.9 2.4AD-392880 21.9 2 83.2 3 10.9 1.5 32.7 3.3 AD-392930 31.4 2.5 95.8 2 9.92.4 42.2 6 AD-392931 75.2 7.7 98.4 4.5 44.3 4.1 108.6 12.5 AD-39293234.7 5.5 99.6 4.9 12.2 0.8 54.5 5.1 AD-392869 21.4 1.8 92.5 12.4 6.9 1.629 2 AD-392870 22.1 3.8 86 13.5 9 1.2 20.7 1.6 AD-392896 50.7 6.7 112.88.3 21.9 3 75.9 9.4 AD-392787 100.4 6.1 114.6 11.3 54.7 3.4 61.6 28.7AD-392720 61.7 30 87.6 4.6 6.6 0.2 34.6 4 AD-392746 54.4 23.1 102.1 22.95.7 0.7 59 6.3 AD-392773 101.8 22 97.6 6.3 30.3 1.5 97.4 6 AD-392807 563.3 76 4.9 11.2 1.4 64.2 4.3 AD-392730 53.3 8.2 102.8 22.2 28.4 1.9 91.95.4 AD-392721 43.9 21.8 93.3 6.7 7.4 0.1 58.1 1.5 AD-392933 51.7 6.288.8 3.3 22 4.4 86.3 7.8 AD-392934 71.4 7.1 100.9 5.6 53.7 3.7 100.1 14AD-392881 34.6 2 104.5 1.5 11 3.9 55 11 AD-392897 47.9 5 103.3 2.7 19.21.9 91.7 7.3 AD-392898 24.7 4.3 98.9 6.7 11.6 2.4 76.1 11.5 AD-39270879.7 6.2 99.5 3.8 57.8 2.5 95.7 6.3 AD-392899 20.7 3.4 75 4.2 12.6 357.9 5.9 AD-392935 25.8 2.6 85.8 2.4 9.5 1.6 44 8 AD-392882 47.9 2 101.94.4 15.9 2.3 77.6 9.3 AD-392738 43.3 10.3 98.8 7.3 9.7 1.4 88 4AD-392739 42.8 13.3 124.4 28 16.6 0.8 82.1 4.9 AD-392936 26.9 3.9 91.32.5 11.7 0.6 45.7 11.4 AD-392900 36.6 1.9 96.1 5.9 11.7 1.5 64.5 4.1AD-392901 49 0.9 106.8 6.4 46.2 4.3 81.8 7.1 AD-392937 36.7 2.7 89.6 312.4 1.4 53 7.9 AD-392883 30.8 2.2 96.6 4.4 8.5 1.2 55.2 3.5 AD-392975112.8 2.1 106.9 2.2 27.9 1.3 95.3 5.5 AD-392938 33 7.9 88.1 3.2 13.2 2.961.6 6.9 AD-392755 100.8 38 105.8 17.6 38.6 2.2 93.2 5.9 AD-392939 36.88 96.2 4.8 9.8 3 59.1 9.1 AD-392940 81.3 12.4 97 3.1 84.6 8.1 93.8 7.5AD-392756 101.7 14.9 94.9 5.7 43.2 4.2 98.9 11.3 AD-392774 99.6 34.897.3 2.2 87.9 3.8 98.6 7.7 AD-392850 89.3 3.3 95.3 4.2 37.4 3.2 102.211.6 AD-392852 91.8 4.8 88.2 5.9 59.9 5.6 103.8 8.7 AD-392830 89.2 1.983.6 9.6 68.2 2.5 89.6 3.7 AD-392808 44.2 17.6 76.1 6.5 9.1 1.1 67.3 3.3AD-392793 72 2.1 84.9 2 33 3.4 68 19.8 AD-392757 71.3 28.8 98.5 1.7 24.41 87.5 5.9 AD-392747 86.8 27.4 99.9 7.2 33.1 0.9 97.6 4.3 AD-392902 29.33.3 134.2 36.1 17.9 1.7 87 6.6 AD-392941 36.9 13.1 82.5 5.4 13.3 1.470.6 13.1 AD-392942 22 3.6 89.2 5.2 6.5 0.8 56.2 4.4 AD-392943 28 4.295.1 4.5 11.3 1.5 57 6.2 AD-392944 27.9 3.5 85.8 4.4 12.9 0.6 53.4 4.1AD-392903 16.4 1 76.8 2.7 7.9 1.1 29 9.1 AD-392775 61.4 30.1 91.8 4.8 150.7 85.1 5.4 AD-392758 53.8 35.1 83.4 8 11.1 0.9 51.6 6.6 AD-392945 33.34.7 101.9 4.9 10.6 1 76.2 3.7 AD-392946 71 6.7 99.6 3.1 39.7 2 90.3 4.5AD-392884 30.2 1.9 90.5 8 10.8 2.3 53.3 2.9 AD-392947 51.8 6 95.8 1.812.4 0.7 68.4 3 AD-392748 84.5 29.8 114 35.3 27.9 1.8 92.7 18.9AD-392759 87.4 36.2 96.7 8.4 22.7 2.7 97 7.7 AD-392837 37.8 0.6 91.9 4.67.9 2.4 36.9 1.6 AD-392970 84.2 7.5 93.4 4.1 7.5 1 41.3 4.2 AD-392976112.8 16.8 112.7 5.3 19.8 1.4 84.1 1.7 AD-392965 82.2 14.1 96.1 5.9 8.21 54.3 1.8 AD-392831 87.9 4.2 82 12.3 12.6 2.8 55.5 5.9 AD-392904 74.22.8 105.9 7.1 26 3 102.4 14.2 AD-392885 30.3 3.2 82.9 6 5.5 1.6 29.9 3.8AD-392886 26.6 3.3 87.3 2.6 9.7 2.2 40.1 4.5 AD-392776 60.2 17 95.7 8.69.4 1.5 69.4 6.5 AD-392887 20.8 3.3 102.3 11.9 8.1 2 34.5 4.7 AD-39272268.7 26.2 95.3 4.1 12.3 2.1 73.8 2.3 AD-392740 93.3 22.3 94.2 5.3 50.72.6 100.4 8.3 AD-392760 68.1 23 96.7 5.6 8.5 0.5 57.3 5.9 AD-392731 39.810.9 99.6 12.7 4.5 2.5 41.1 11.1 AD-392709 74.4 24.7 107.4 13.6 11.8 0.778.2 5.3 AD-392723 58.8 23.6 119.7 22.1 14.2 3.1 72.1 3.9 AD-392948 32.87.4 84.3 2.3 6.5 0.5 33 2.3 AD-392724 59.7 13.5 93.8 7.2 13 1.6 58.3 5.5AD-392949 49 2.8 92.9 2 15.8 1.7 70.8 2.6 AD-392725 40.2 6.5 95.7 5.9 102.8 54.4 2.5 AD-392950 25.1 4.1 83.7 5.5 8.2 0.9 50.2 3.9 AD-392732 27.65.2 92.4 16.7 7.4 1.1 30.6 1.5 AD-392726 57.8 9.3 96 4.8 9 0.6 70 5.9AD-392733 79.3 18 92.3 5 40.3 1.9 96.6 7.5 AD-392906 75.4 3.6 104.5 2.137.2 4 107 18.3 AD-392862 33.1 2.3 84.5 4.2 10.7 2 54 5.2 AD-392951 416.5 94.1 8 13.4 0.5 70.5 4.1 AD-392871 46.6 11.3 95.8 14.3 12.2 1.8 35.75.2 AD-392872 69.6 11 92 7.4 17.5 3 55.4 6.7 AD-392952 74.8 6.9 101.15.8 73 4.1 94.5 4.3 AD-392907 74.8 4.4 99.4 4.5 71.4 6.2 102.2 16.2AD-392953 79.5 5.3 101.7 4.3 72.4 3.5 90.6 3.7 AD-392741 85 16.2 93.14.3 90.3 5.6 97 5.7 AD-392908 71.7 5 105.4 2.3 72.2 1.6 95 9 AD-39297793.7 7.9 111.3 3.2 68 2.7 80.1 2.5 AD-392847 92.1 1.9 97.9 1.8 82.4 585.7 8.1 AD-392809 93.5 7 93.9 10 81.9 7.5 83.3 5.7 AD-392810 93 6.188.8 5.9 76.9 5.4 90.6 2.9 AD-392777 88.2 20.1 92.7 7.2 86 5 101.9 13AD-392960 85 8.7 103.7 8.7 73 3.8 87 6 AD-392873 95.5 2.9 95.5 5.6 76.43.7 49.1 15.4 AD-392889 64.1 5.5 126.2 36.5 71.1 4.8 85.6 7.4 AD-39295468.9 7.2 98.1 6 66.7 3.5 75.1 4.3 AD-392955 83 3.1 98.6 5.7 73.6 1.388.6 2.9 AD-392909 61.4 4.4 101.1 5.8 67.3 4.7 85.8 10.4 AD-392710 11029.8 165.2 53.6 66.7 3.8 86 9.1 AD-392956 71.5 9.3 93.1 3.8 63.5 4.578.9 2.7 AD-392874 77.2 2.9 98.8 4.1 67.5 9.5 64.9 15.1 AD-392957 59.510.6 98.9 19 60.5 4.8 72.4 2 AD-392958 80.4 5.5 95.9 8.2 83.3 5 102.96.3 AD-392959 67.6 6.5 99 6.1 75.9 3.1 89.4 3.3 AD-392788 106.7 6 111.99.1 92.1 4 87.4 6.6

Certain groups of agents were identified as residing in regions ofparticularly efficacious APP knockdown targeting. As shown in the aboveresults, some regions of the APP transcript appear to be relatively moresusceptible to targeting with RNAi agents of the disclosure than otherregions—e.g., the agents that target APP positions 2639 to 2689 in theNM_000484 sequence (i.e., RNAi agents AD-392785, AD-392829, AD-392920,AD-392921, AD-392768, AD-392805, AD-392769, AD-392753, AD-392714,AD-392703 and AD-392715) exhibited particularly robust knockdown resultsin the Be(2)C cell line, suggesting a possible “hotspot”, with likelysimilar activity of other, overlapping RNAi agents targeting thesepositions of the APP transcript. It is therefore expressly contemplatedthat any RNAi agents possessing target sequences that reside fullywithin the following windows of NM_000484 positions are likely toexhibit robust APP inhibitory effect: APP NM_00484 positions 1891-1919;APP NM_00484 positions 2282-2306; APP NM_00484 positions 2464-2494; APPNM_00484 positions 2475-2638; APP NM_00484 positions 2621-2689; APPNM_00484 positions 2682-2725; APP NM_00484 positions 2705-2746; APPNM_00484 positions 2726-2771; APP NM_00484 positions 2754-2788; APPNM_00484 positions 2782-2813; APP NM_00484 positions 2801-2826; APPNM_00484 positions 2847-2890; APP NM_00484 positions 2871-2896; APPNM_00484 positions 2882-2960; APP NM_00484 positions 2942-2971; APPNM_00484 positions 2951-3057; APP NM_00484 positions 3172-3223; APPNM_00484 positions 3209-3235; NM_00484 positions 3256-3289; NM_00484positions 3302-3338; APP NM_00484 positions 3318-3353; and APP NM_00484positions 3334-3361.

TABLE 5A Mouse APP Modified Sequences SEQ SEQ SEQ Sense Sequence IDAntisense Sequence ID ID Duplex Name (5′ to 3′) NO (5′ to 3′) NOmRNA target sequence NO AD-397175 csasugu(Uhd)CfuGf 1403VPusUfsgagUfuUfAfccacAfgAfacaugsgsc 1404 GCCAUGUUCUGUGGUAAACUCAA 1405UfGfguaaacucaaL96 AD-397176 usgsuuc(Uhd)GfuGf 1406VPusGfsuugAfgUfUfuaccAfcAfgaacasusg 1407 CAUGUUCUGUGGUAAACUCAACA 1408GfUfaaacucaacaL96 AD-397177 asusguu(Chd)UfgUf 1409VPusUfsugaGfuULUfaccaCfaGfaacausgsg 1410 CCAUGUUCUGUGGUAAACUCAAC 1411GfGfuaaacucaaaL96 AD-397178 csusgug(Ghd)UfaAf 1412VPusGfscauGfuUfGfaguuUfaCfcacagsasa 1413 UUCUGUGGUAAACUCAACAUGCA 1414AfCfucaacaugcaL96 AD-397179 gsgsuaa(Ahd)CfuCf 1415VPusAfsuguGfcAfUfguugAfgUfuuaccsasc 1416 GUGGUAAACUCAACAUGCACAUG 1417AfAfcaugcacauaL96 AD-397180 usgsugg(Uhd)AfaAf 1418VPusUfsgcaUfgUfUfgaguUfuAfccacasgsa 1419 UCUGUGGUAAACUCAACAUGCAC 1420CfUfcaacaugcaaL96 AD-397181 gsasaga(Ghd)CfaCf 1421VPusCfsgugCfaAfGfuuagUfgCfucuucsusc 1422 GAGAAGAGCACUAACUUGCACGA 1423UfAfacuugcacgaL96 AD-397182 cscsgcu(Ghd)GfuAf 1424VPusUfsgacAfuCfAfaaguAfcCfagcggsgsa 1425 UCCCGCUGGUACUUUGAUGUCAC 1426CfUfuugaugucaaL96 AD-397183 cscsaug(Uhd)UfcUf 1427VPusGfsaguUfuAfCfcacaGfaAfcauggscsg 1428 CGCCAUGUUCUGUGGUAAACUCA 1429GfUfgguaaacucaL96 AD-397184 gsusggu(Ahd)AfaCf 1430VPusGfsugcAfuGfUfugagUfuUfaccacsasg 1431 CUGUGGUAAACUCAACAUGCACA 1432UfCfaacaugcacaL96 AD-397185 gsasacu(Ghd)CfaGf 1433VPusAfscguUfuGfUfgaucUfgCfaguucsasg 1434 CUGAACUGCAGAUCACAAACGUG 1435AfUfcacaaacguaL96 AD-397186 asasgag(Chd)AfcUf 1436VPusUfscguGfcAfAfguuaGfuGfcucuuscsu 1437 AGAAGAGCACUAACUUGCACGAC 1438AfAfcuugcacgaaL96 AD-397187 asgscac(Uhd)AfaCf 1439VPusUfsaguCfgUfGfcaagUfuAfgugcuscsu 1440 AGAGCACUAACUUGCACGACUAU 1441UfUfgcacgacuaaL96 AD-397188 gscsacu(Ahd)AfcUf 1442VPusAfsuagUfcGfUfgcaaGfuUfagugcsusc 1443 GAGCACUAACUUGCACGACUAUG 1444UfGfcacgacuauaL96 AD-397189 asasagu(Uhd)UfaCf 1445VPusGfsguaGfuCfUfugagUfaAfacuuusgsg 1446 CCAAAGUUUACUCAAGACUACCA 1447UfCfaagacuaccaL96 AD-397190 csgscau(Ghd)AfaCf 1448VPusGfsacaGfaGfAfcuggUfuCfaugcgscsu 1449 AGCGCAUGAACCAGUCUCUGUCC 1450CfAfgucucugucaL96 AD-397191 csascau(Chd)GfuGf 1451VPusCfsgguAfaGfGfaaucAfcGfaugugsgsg 1452 CCCACAUCGUGAUUCCUUACCGU 1453AfUfuccuuaccgaL96 AD-397192 asusgcu(Ghd)AfaGf 1454VPusCfsggaCfgUfAfcuucUfuCfagcausgsu 1455 ACAUGCUGAAGAAGUACGUCCGU 1456AfAfguacguccgaL96 AD-397193 gsasgcg(Chd)AfuGf 1457VPusAfsgagAfcUfGfguucAfuGfcgcucsgsu 1458 ACGAGCGCAUGAACCAGUCUCUG 1459AfAfccagucucuaL96 AD-397194 gsasgca(Ghd)AfaCf 1460VPusUfscguCfgGfAfguagUfuCfugcucscsu 1461 AGGAGCAGAACUACUCCGACGAU 1462UfAfcuccgacgaaL96 AD-397195 csasccc(Ahd)CfaUf 1463VPusAfsaggAfaUfCfacgaUfgUfgggugsusg 1464 CACACCCACAUCGUGAUUCCUUA 1465CfGfugauuccuuaL96 AD-397196 asgsagc(Ahd)CfuAf 1466VPusGfsucgUfgCfAfaguuAfgUfgcucususc 1467 GAAGAGCACUAACUUGCACGACU 1468AfCfuugcacgacaL96 AD-397197 csascua(Ahd)CfuUf 1469VPusCfsauaGfuCfGfugcaAfgUfuagugscsu 1470 AGCACUAACUUGCACGACUAUGG 1471GfCfacgacuaugaL96 AD-397198 csuscaa(Ghd)AfcUf 1472VPusGfsguuCfaCfUfgguaGfuCfuugagsusa 1473 UACUCAAGACUACCAGUGAACCU 1474AfCfcagugaaccaL96 AD-397199 asgscac(Ahd)CfcCf 1475VPusAfsaaaUfgCfUfuuagGfgUfgugcusgsu 1476 ACAGCACACCCUAAAGCAUUUUG 1477UfAfaagcauuuuaL96 AD-397200 asasgga(Ghd)CfaGf 1478VPusUfscggAfgUfAfguucUfgCfuccuuscsu 1479 AGAAGGAGCAGAACUACUCCGAC 1480AfAfcuacuccgaaL96 AD-397201 gsgsagc(Ahd)GfaAf 1481VPusCfsgucGfgAfGfuagLfacUfgcuccsusu 1482 AAGGAGCAGAACUACUCCGACGA 1483CfUfacuccgacgaL96 AD-397202 gsasaac(Ahd)GfuAf 1484VPusGfsgauGfgAfUfguguAfcUfguuucsusu 1485 AAGAAACAGUACACAUCCAUCCA 1486CfAfcauccauccaL96 AD-397203 csusgaa(Chd)UfgCf 1487VPusGfsuuuGfuGfAfucugCfaGfuucagsgsg 1488 CCCUGAACUGCAGAUCACAAACG 1489AfGfaucacaaacaL96 AD-397204 cscsaca(Uhd)CfgUf 1490VPusGfsguaAfgGfAfaucaCfgAfuguggsgsu 1491 ACCCACAUCGUGAUUCCUUACCG 1492GfAfuuccuuaccaL96 AD-397205 gsusgcc(Chd)GfaCf 1493VPusAfsacuUfgCfAfcuugUfcGfggcacsgsa 1494 UCGUGCCCGACAAGUGCAAGUUC 1495AfAfgugcaaguuaL96 AD-397206 gsascua(Chd)CfaGf 1496VPusGfsaagAfgGfUfucacUfgGfuagucsusu 1497 AAGACUACCAGUGAACCUCUUCC 1498UfGfaaccucuucaL96 AD-397207 gsusccg(Chd)CfaUf 1499VPusAfsccaGfuUfUfuugaUfgGfcggacsusu 1500 AAGUCCGCCAUCAAAAACUGGUG 1501CfAfaaaacugguaL96 AD-397208 gsgsccc(Uhd)CfgAf 1502VPusUfsgauGfuAfAfuucuCfgAfgggccsasg 1503 CUGGCCCUCGAGAAUUACAUCAC 1504GfAfauuacaucaaL96 AD-397209 csasugc(Uhd)GfaAf 1505VPusGfsgacGfuAfCfuucuUfcAfgcaugsusu 1506 AACAUGCUGAAGAAGUACGUCCG 1507GfAfaguacguccaL96 AD-397210 usgscug(Ahd)AfgAf 1508VPusAfscggAfcGfUfacuuCfuUfcagcasusg 1509 CAUGCUGAAGAAGUACGUCCGUG 1510AfGfuacguccguaL96 AD-397211 uscscgc(Chd)AfuCf 1511VPusCfsaccAfgUfUfuuugAfuGfgcggascsu 1512 AGUCCGCCAUCAAAAACUGGUGU 1513AfAfaaacuggugaL96 AD-397212 ususgca(Chd)GfaCf 1514VPusAfsgcaUfgCfCfauagUfcGfugcaasgsu 1515 ACUUGCACGACUAUGGCAUGCUG 1516UfAfuggcaugcuaL96 AD-397213 uscscca(Ghd)GfuCf 1517VPusCfsauuCfuCfUfcaugAfcCfugggascsa 1518 UGUCCCAGGUCAUGAGAGAAUGG 1519AfUfgagagaaugaL96 AD-397214 csusgaa(Ghd)AfaGf 1520VPusGfscacGfgAfCfguacUfuCfuucagscsa 1521 UGCUGAAGAAGUACGUCCGUGCG 1522UfAfcguccgugcaL96 AD-397215 csgsugu(Ghd)AfuCf 1523VPusAfsugcGfcUfCfguagAfuCfacacgsgsa 1524 UCCGUGUGAUCUACGAGCGCAUG 1525UfAfcgagcgcauaL96 AD-397216 usascug(Chd)CfaAf 1526VPusGfsgguAfgAfCfcucuUfgGfcaguascsu 1527 AGUACUGCCAAGAGGUCUACCCU 1528GfAfggucuacccaL96 AD-397217 csasccg(Ahd)GfaGf 1529VPusUfsgggAfcAfUfucucUfcUfcggugscsu 1530 AGCACCGAGAGAGAAUGUCCCAG 1531AfGfaaugucccaaL96 AD-397218 csasagg(Chd)CfuCf 1532VPusGfsaacAfcAfUfgaugAfgGfccuugsgsg 1533 CCCAAGGCCUCAUCAUGUGUUCA 1534AfUfcauguguucaL96 AD-397219 gscsuga(Ahd)GfaAf 1535VPusCfsacgGfaCfGfuacuUfcUfucagcsasu 1536 AUGCUGAAGAAGUACGUCCGUGC 1537GfUfacguccgugaL96 AD-397220 asasgca(Uhd)UfuUf 1538VPusCfsgcaCfaUfGfuucaAfaAfugcuususa 1539 UAAAGCAUUUUGAACAUGUGCGC 1540GfAfacaugugcgaL96 AD-397221 csasccu(Chd)CfgUf 1541VPusUfscguAfgAfUfcacaCfgGfaggugsusg 1542 CACACCUCCGUGUGAUCUACGAG 1543GfUfgaucuacgaaL96 AD-397222 gsasagg(Ahd)GfcAf 1544VPusCfsggaGfuAfGfuucuGfcUfccuucsusg 1545 CAGAAGGAGCAGAACUACUCCGA 1546GfAfacuacuccgaL96 AD-397223 gsasaga(Ahd)AfcAf 1547VPusUfsggaUfgUfGfuacuGfuUfucuucsusu 1548 AAGAAGAAACAGUACACAUCCAU 1549GfUfacacauccaaL96 AD-397224 gsusacu(Ghd)CfcAf 1550VPusGfsguaGfaCfCfucuuGfgCfaguacsusg 1551 CAGUACUGCCAAGAGGUCUACCC 1552AfGfaggucuaccaL96 AD-397225 ascsugc(Chd)AfaGf 1553VPusAfsgggUfaGfAfccucUfuGfgcagusasc 1554 GUACUGCCAAGAGGUCUACCCUG 1555AfGfgucuacccuaL96 AD-397226 ascsuaa(Chd)UfuGf 1556VPusCfscauAfgUfCfgugcAfaGfuuagusgsc 1557 GCACUAACUUGCACGACUAUGGC 1558CfAfcgacuauggaL96 AD-397227 gsusccc(Ahd)UfuCf 1559VPusCfscgcCfgUfAfaaagAfaUfgggacsasc 1560 GUGUCCCAUUCUUUUACGGCGGA 1561UfUfuuacggcggaL96 AD-397228 asasgcu(Ghd)AfcAf 1562VPusAfsacgGfcCfUfucuuGfuCfagcuususg 1563 CAAAGCUGACAAGAAGGCCGUUA 1564AfGfaaggccguuaL96 AD-397229 usgsaca(Ahd)GfaAf 1565VPusGfsgauAfaCfGfgccuUfcUfugucasgsc 1566 GCUGACAAGAAGGCCGUUAUCCA 1567GfGfccguuauccaL96 AD-397230 asgscau(Uhd)UfuGf 1568VPusGfscgcAfcAfUfguucAfaAfaugcususu 1569 AAAGCAUUUUGAACAUGUGCGCA 1570AfAfcaugugcgcaL96 AD-397231 usgsuga(Uhd)CfuAf 1571VPusUfscauGfcGfCfucguAfgAfucacascsg 1572 CGUGUGAUCUACGAGCGCAUGAA 1573CfGfagcgcaugaaL96 AD-397233 csasgcg(Ahd)GfaAf 1574VPusAfsguuAfgUfGfcucuUfcUfcgcugscsa 1575 UGCAGCGAGAAGAGCACUAACUU 1576GfAfgcacuaacuaL96 AD-397234 asgscgu(Ghd)UfcAf 1577VPusAfsaacUfuUfGfgguuGfaCfacgcusgsc 1578 GCAGCGUGUCAACCCAAAGUUUA 1579AfCfccaaaguuuaL96 AD-397235 usgsuca(Ahd)CfcCf 1580VPusGfsaguAfaAfCfuuugGfgUfugacascsg 1581 CGUGUCAACCCAAAGUUUACUCA 1582AfAfaguuuacucaL96 AD-397236 usgsucc(Chd)AfuUf 1583VPusCfsgccGfuAfAfaagaAfuGfggacascsa 1584 UGUGUCCCAUUCUUUUACGGCGG 1585CfUfuuuacggcgaL96 AD-397237 gsusguc(Ahd)AfcCf 1586VPusAfsguaAfaCfUfuuggGfuUfgacacsgsc 1587 GCGUGUCAACCCAAAGUUUACUC 1588CfAfaaguuuacuaL96 AD-397238 asasgau(Chd)CfuGf 1589VPusGfsggaAfgUfUfuaucAfgGfaucuusgsg 1590 CCAAGAUCCUGAUAAACUUCCCA 1591AfUfaaacuucccaL96 AD-397239 asgsauc(Chd)UfgAf 1592VPusUfsgggAfaGfUfuuauCfaGfgaucususg 1593 CAAGAUCCUGAUAAACUUCCCAC 1594UfAfaacuucccaaL96 AD-397240 csusuac(Chd)GfuUf 1595VPusAfsccaAfcUfAfggcaAfcGfguaagsgsa 1596 UCCUUACCGUUGCCUAGUUGGUG 1597GfCfcuaguugguaL96 AD-397241 gsusgug(Uhd)CfcCf 1598VPusCfsguaAfaAfGfaaugGfgAfcacacsusu 1599 AAGUGUGUCCCAUUCUUUUACGG 1600AfUfucuuuuacgaL96 AD-397242 gsusguc(Chd)CfaUf 1601VPusGfsccgUfaAfAfagaaUfgGfgacacsasc 1602 GUGUGUCCCAUUCUUUUACGGCG 1603UfCfuuuuacggcaL96 AD-397243 csasuag(Chd)AfaCf 1604VPusUfsgacAfaUfCfacggUfuGfcuaugsasc 1605 GUCAUAGCAACCGUGAUUGUCAU 1606CfGfugauugucaaL96 AD-397244 gsasacg(Ghd)AfuAf 1607VPusUfsuggAfuUfCfucauAfuCfcguucsusg 1608 CAGAACGGAUAUGAGAAUCCAAC 1609UfGfagaauccaaaL96 AD-397245 usgsugu(Chd)CfcAf 1610VPusCfscguAfaAfAfgaauGfgGfacacascsu 1611 AGUGUGUCCCAUUCUUUUACGGC 1612UfUfcuuuuacggaL96 AD-397246 gscsaac(Chd)GfuGf 1613VPusGfsugaUfgAfCfaaucAfcGfguugcsusa 1614 UAGCAACCGUGAUUGUCAUCACC 1615AfUfugucaucacaL96 AD-397247 gscsagc(Ghd)AfgAf 1616VPusGfsuuaGfuGfCfucuuCfuCfgcugcsasu 1617 AUGCAGCGAGAAGAGCACUAACU 1618AfGfagcacuaacaL96 AD-397248 csasgaa(Uhd)UfcGf 1619VPusUfsgaaUfcAfUfguccGfaAfuucugscsa 1620 UGCAGAAUUCGGACAUGAUUCAG 1621GfAfcaugauucaaL96 AD-397249 uscscug(Ahd)UfaAf 1622VPusUfscguGfgGfAfaguuUfaUfcaggasusc 1623 GAUCCUGAUAAACUUCCCACGAC 1624AfCfuucccacgaaL96 AD-397250 asgsaac(Ghd)GfaUf 1625VPusUfsggaUfuCfUfcauaUfcCfguucusgsc 1626 GCAGAACGGAUAUGAGAAUCCAA 1627AfUfgagaauccaaL96 AD-397251 cscsuua(Chd)CfgUf 1628VPusCfscaaCfuAfGfgcaaCfgGfuaaggsasa 1629 UUCCUUACCGUUGCCUAGUUGGU 1630UfGfccuaguuggaL96 AD-397252 asusccu(Ghd)AfuAf 1631VPusCfsgugGfgAfAfguuuAfuCfaggauscsu 1632 AGAUCCUGAUAAACUUCCCACGA 1633AfAfcuucccacgaL96 AD-397253 cscsuga(Uhd)AfaAf 1634VPusGfsucgUfgGfGfaaguUfuAfucaggsasu 1635 AUCCUGAUAAACUUCCCACGACA 1636CfUfucccacgacaL96 AD-397254 csgsgau(Ghd)GfaUf 1637VPusGfsucuCfaCfAfaacaUfcCfauccgscsu 1638 AGCGGAUGGAUGUUUGUGAGACC 1639GfUfuugugagacaL96 AD-397255 gsascac(Ghd)GfaAf 1640VPusAfsugcAfgUfAfcucuUfcCfgugucsasa 1641 UUGACACGGAAGAGUACUGCAUG 1642GfAfguacugcauaL96 AD-397256 gscsagc(Ahd)GfaAf 1643VPusUfscucAfuAfUfccguUfcUfgcugcsasu 1644 AUGCAGCAGAACGGAUAUGAGAA 1645CfGfgauaugagaaL96 AD-397257 gscsaga(Ahd)CfgGf 1646VPusGfsauuCfuCfAfuaucCfgUfucugcsusg 1647 CAGCAGAACGGAUAUGAGAAUCC 1648AfUfaugagaaucaL96 AD-397258 csasgaa(Chd)GfgAf 1649VPusGfsgauUfcUfCfauauCfcGfuucugscsu 1650 AGCAGAACGGAUAUGAGAAUCCA 1651UfAfugagaauccaL96 AD-397259 ascscgu(Chd)GfcCf 1652VPusCfsaugUfcUfCfuuugGfcGfacggusgsu 1653 ACACCGUCGCCAAAGAGACAUGC 1654AfAfagagacaugaL96 AD-397260 gsusucu(Ghd)UfgGf 1655VPusUfsguuGfaGfUfuuacCfaCfagaacsasu 1656 AUGUUCUGUGGUAAACUCAACAU 1657UfAfaacucaacaaL96 AD-397261 gsgsuac(Uhd)UfuGf 1658VPusUfsucaGfuGfAfcaucAfaAfguaccsasg 1659 CUGGUACUUUGAUGUCACUGAAG 1660AfUfgucacugaaaL96 AD-397262 cscscaa(Ahd)GfuUf 1661VPusAfsgucUfuGfAfguaaAfcUfuugggsusu 1662 AACCCAAAGUUUACUCAAGACUA 1663UfAfcucaagacuaL96 AD-397263 cscsaaa(Ghd)UfuUf 1664VPusUfsaguCfuUfGfaguaAfaCfuuuggsgsu 1665 ACCCAAAGUUUACUCAAGACUAC 1666AfCfucaagacuaaL96 AD-397264 csasuca(Uhd)GfuGf 1667VPusAfsgcaUfgUfUfgaacAfcAfugaugsasg 1668 CUCAUCAUGUGUUCAACAUGCUG 1669UfUfcaacaugcuaL96 AD-397265 asascau(Ghd)CfuGf 1670VPusAfscguAfcUfUfcuucAfgCfauguusgsa 1671 UCAACAUGCUGAAGAAGUACGUC 1672AfAfgaaguacguaL96 AD-397266 ususcug(Uhd)GfgUf 1673VPusAfsuguUfgAfGfuuuaCfcAfcagaascsa 1674 UGUUCUGUGGUAAACUCAACAUG 1675AfAfacucaacauaL96 AD-397267 uscsugu(Ghd)GfuAf 1676VPusCfsaugUfuGfAfguuuAfcCfacagasasc 1677 GUUCUGUGGUAAACUCAACAUGC 1678AfAfcucaacaugaL96

TABLE 5BMouse APP Modified Sequences, No “L96” Linker, No Vinyl-Phosphate SEQSEQ SEQ Sense Sequence ID ID ID Duplex Name (5′ to 3′) NOAntisense Sequence (5′ to 3′) NO mRNA target sequence NO AD-397175csasugu(Uhd)CfuGf 1403 usUfsgagUfuUfAfccacAfgAfacaugsgsc 1404GCCAUGUUCUGUGGUAAACUCAA 1405 UfGfguaaacucaa AD-397176 usgsuuc(Uhd)GfuGf1406 usGfsuugAfgUfUfuaccAfcAfgaacasusg 1407 CAUGUUCUGUGGUAAACUCAACA 1408GfUfaaacucaaca AD-397177 asusguu(Chd)UfgUf 1409usUfsugaGfuUfUfaccaCfaGfaacausgsg 1410 CCAUGUUCUGUGGUAAACUCAAC 1411GfGfuaaacucaaa AD-397178 csusgug(Ghd)UfaAf 1412usGfscauGfuUfGfaguuUfaCfcacagsasa 1413 UUCUGUGGUAAACUCAACAUGCA 1414AfCfucaacaugca AD-397179 gsgsuaa(Ahd)CfuCf 1415usAfsuguGfcAfUfguugAfgUfuuaccsasc 1416 GUGGUAAACUCAACAUGCACAUG 1417AfAfcaugcacaua AD-397180 usgsugg(Uhd)AfaAf 1418usUfsgcaUfgUfUfgaguUfuAfccacasgsa 1419 UCUGUGGUAAACUCAACAUGCAC 1420CfUfcaacaugcaa AD-397181 gsasaga(Ghd)CfaCf 1421usCfsgugCfaAfGfuuagUfgCfucuucsusc 1422 GAGAAGAGCACUAACUUGCACGA 1423UfAfacuugcacga AD-397182 cscsgcu(Ghd)GfuAf 1424usUfsgacAfuCfAfaaguAfcCfagcggsgsa 1425 UCCCGCUGGUACUUUGAUGUCAC 1426CfUfuugaugucaa AD-397183 cscsaug(Uhd)UfcUf 1427usGfsaguUfuAfCfcacaGfaAfcauggscsg 1428 CGCCAUGUUCUGUGGUAAACUCA 1429GfUfgguaaacuca AD-397184 gsusggu(Ahd)AfaCf 1430usGfsugcAfuGfUfugagUfuUfaccacsasg 1431 CUGUGGUAAACUCAACAUGCACA 1432UfCfaacaugcaca AD-397185 gsasacu(Ghd)CfaGf 1433usAfscguUfuGfUfgaucUfgCfaguucsasg 1434 CUGAACUGCAGAUCACAAACGUG 1435AfUfcacaaacgua AD-397186 asasgag(Chd)AfcUf 1436usUfscguGfcAfAfguuaGfuGfcucuuscsu 1437 AGAAGAGCACUAACUUGCACGAC 1438AfAfcuugcacgaa AD-397187 asgscac(Uhd)AfaCf 1439usUfsaguCfgUfGfcaagUfuAfgugcuscsu 1440 AGAGCACUAACUUGCACGACUAU 1441UfUfgcacgacuaa AD-397188 gscsacu(Ahd)AfcUf 1442usAfsuagUfcGfUfgcaaGfuUfagugcsusc 1443 GAGCACUAACUUGCACGACUAUG 1444UfGfcacgacuaua AD-397189 asasagu(Uhd)UfaCf 1445usGfsguaGfuCfUfugagUfaAfacuuusgsg 1446 CCAAAGUUUACUCAAGACUACCA 1447UfCfaagacuacca AD-397190 csgscau(Ghd)AfaCf 1448usGfsacaGfaGfAfcuggUfuCfaugcgscsu 1449 AGCGCAUGAACCAGUCUCUGUCC 1450CfAfgucucuguca AD-397191 csascau(Chd)GfuGf 1451usCfsgguAfaGfGfaaucAfcGfaugugsgsg 1452 CCCACAUCGUGAUUCCUUACCGU 1453AfUfuccuuaccga AD-397192 asusgcu(Ghd)AfaGf 1454usCfsggaCfgUfAfcuucUfuCfagcausgsu 1455 ACAUGCUGAAGAAGUACGUCCGU 1456AfAfguacguccga AD-397193 gsasgcg(Chd)AfuGf 1457usAfsgagAfcUfGfguucAfuGfcgcucsgsu 1458 ACGAGCGCAUGAACCAGUCUCUG 1459AfAfccagucucua AD-397194 gsasgca(Ghd)AfaCf 1460usUfscguCfgGfAfguagUfuCfugcucscsu 1461 AGGAGCAGAACUACUCCGACGAU 1462UfAfcuccgacgaa AD-397195 csasccc(Ahd)CfaUf 1463usAfsaggAfaUfCfacgaUfgUfgggugsusg 1464 CACACCCACAUCGUGAUUCCUUA 1465CfGfugauuccuua AD-397196 asgsagc(Ahd)CfuAf 1466usGfsucgUfgCfAfaguuAfgUfgcucususc 1467 GAAGAGCACUAACUUGCACGACU 1468AfCfuugcacgaca AD-397197 csascua(Ahd)CfuUf 1469usCfsauaGfuCfGfugcaAfgUfuagugscsu 1470 AGCACUAACUUGCACGACUAUGG 1471GfCfacgacuauga AD-397198 csuscaa(Ghd)AfcUf 1472usGfsguuCfaCfUfgguaGfuCfuugagsusa 1473 UACUCAAGACUACCAGUGAACCU 1474AfCfcagugaacca AD-397199 asgscac(Ahd)CfcCf 1475usAfsaaaUfgCfUfuuagGfgUfgugcusgsu 1476 ACAGCACACCCUAAAGCAUUUUG 1477UfAfaagcauuuua AD-397200 asasgga(Ghd)CfaGf 1478usUfscggAfgUfAfguucUfgCfuccuuscsu 1479 AGAAGGAGCAGAACUACUCCGAC 1480AfAfcuacuccgaa AD-397201 gsgsagc(Ahd)GfaAf 1481usCfsgucGfgAfGfuaguUfcUfgcuccsusu 1482 AAGGAGCAGAACUACUCCGACGA 1483CfUfacuccgacga AD-397202 gsasaac(Ahd)GfuAf 1484usGfsgauGfgAfUfguguAfcUfguuucsusu 1485 AAGAAACAGUACACAUCCAUCCA 1486CfAfcauccaucca AD-397203 csusgaa(Chd)UfgCf 1487usGfsuuuGfuGfAfucugCfaGfuucagsgsg 1488 CCCUGAACUGCAGAUCACAAACG 1489AfGfaucacaaaca AD-397204 cscsaca(Uhd)CfgUf 1490usGfsguaAfgGfAfaucaCfgAfuguggsgsu 1491 ACCCACAUCGUGAUUCCUUACCG 1492GfAfuuccuuacca AD-397205 gsusgcc(Chd)GfaCf 1493usAfsacuUfgCfAfcuugUfcGfggcacsgsa 1494 UCGUGCCCGACAAGUGCAAGUUC 1495AfAfgugcaaguua AD-397206 gsascua(Chd)CfaGf 1496usGfsaagAfgGfUfucacUfgGfuagucsusu 1497 AAGACUACCAGUGAACCUCUUCC 1498UfGfaaccucuuca AD-397207 gsusccg(Chd)CfaUf 1499usAfsccaGfuUfUfuugaUfgGfcggacsusu 1500 AAGUCCGCCAUCAAAAACUGGUG 1501CfAfaaaacuggua AD-397208 gsgsccc(Uhd)CfgAf 1502usUfsgauGfuAfAfuucuCfgAfgggccsasg 1503 CUGGCCCUCGAGAAUUACAUCAC 1504GfAfauuacaucaa AD-397209 csasugc(Uhd)GfaAf 1505usGfsgacGfuAfCfuucuUfcAfgcaugsusu 1506 AACAUGCUGAAGAAGUACGUCCG 1507GfAfaguacgucca AD-397210 usgscug(Ahd)AfgAf 1508usAfscggAfcGfUfacuuCfuUfcagcasusg 1509 CAUGCUGAAGAAGUACGUCCGUG 1510AfGfuacguccgua AD-397211 uscscgc(Chd)AfuCf 1511usCfsaccAfgUfUfuuugAfuGfgcggascsu 1512 AGUCCGCCAUCAAAAACUGGUGU 1513AfAfaaacugguga AD-397212 ususgca(Chd)GfaCf 1514usAfsgcaUfgCfCfauagUfcGfugcaasgsu 1515 ACUUGCACGACUAUGGCAUGCUG 1516UfAfuggcaugcua AD-397213 uscscca(Ghd)GfuCf 1517usCfsauuCfuCfUfcaugAfcCfugggascsa 1518 UGUCCCAGGUCAUGAGAGAAUGG 1519AfUfgagagaauga AD-397214 csusgaa(Ghd)AfaGf 1520usGfscacGfgAfCfguacUfuCfuucagscsa 1521 UGCUGAAGAAGUACGUCCGUGCG 1522UfAfcguccgugca AD-397215 csgsugu(Ghd)AfuCf 1523usAfsugcGfcUfCfguagAfuCfacacgsgsa 1524 UCCGUGUGAUCUACGAGCGCAUG 1525UfAfcgagcgcaua AD-397216 usascug(Chd)CfaAf 1526usGfsgguAfgAfCfcucuUfgGfcaguascsu 1527 AGUACUGCCAAGAGGUCUACCCU 1528GfAfggucuaccca AD-397217 csasccg(Ahd)GfaGf 1529usUfsgggAfcAfUfucucUfcUfcggugscsu 1530 AGCACCGAGAGAGAAUGUCCCAG 1531AfGfaaugucccaa AD-397218 csasagg(Chd)CfuCf 1532usGfsaacAfcAfUfgaugAfgGfccuugsgsg 1533 CCCAAGGCCUCAUCAUGUGUUCA 1534AfUfcauguguuca AD-397219 gscsuga(Ahd)GfaAf 1535usCfsacgGfaCfGfuacuUfcUfucagcsasu 1536 AUGCUGAAGAAGUACGUCCGUGC 1537GfUfacguccguga AD-397220 asasgca(Uhd)UfuUf 1538usCfsgcaCfaUfGfuucaAfaAfugcuususa 1539 UAAAGCAUUUUGAACAUGUGCGC 1540GfAfacaugugcga AD-397221 csasccu(Chd)CfgUf 1541usUfscguAfgAfUfcacaCfgGfaggugsusg 1542 CACACCUCCGUGUGAUCUACGAG 1543GfUfgaucuacgaa AD-397222 gsasagg(Ahd)GfcAf 1544usCfsggaGfuAfGfuucuGfcUfccuucsusg 1545 CAGAAGGAGCAGAACUACUCCGA 1546GfAfacuacuccga AD-397223 gsasaga(Ahd)AfcAf 1547usUfsggaUfgUfGfuacuGfuUfucuucsusu 1548 AAGAAGAAACAGUACACAUCCAU 1549GfUfacacauccaa AD-397224 gsusacu(Ghd)CfcAf 1550usGfsguaGfaCfCfucuuGfgCfaguacsusg 1551 CAGUACUGCCAAGAGGUCUACCC 1552AfGfaggucuacca AD-397225 ascsugc(Chd)AfaGf 1553usAfsgggUfaGfAfccucUfuGfgcagusasc 1554 GUACUGCCAAGAGGUCUACCCUG 1555AfGfgucuacccua AD-397226 ascsuaa(Chd)UfuGf 1556usCfscauAfgUfCfgugcAfaGfuuagusgsc 1557 GCACUAACUUGCACGACUAUGGC 1558CfAfcgacuaugga AD-397227 gsusccc(Ahd)UfuCf 1559usCfscgcCfgUfAfaaagAfaUfgggacsasc 1560 GUGUCCCAUUCUUUUACGGCGGA 1561UfUfuuacggcgga AD-397228 asasgcu(Ghd)AfcAf 1562usAfsacgGfcCfUfucuuGfuCfagcuususg 1563 CAAAGCUGACAAGAAGGCCGUUA 1564AfGfaaggccguua AD-397229 usgsaca(Ahd)GfaAf 1565usGfsgauAfaCfGfgccuUfcUfugucasgsc 1566 GCUGACAAGAAGGCCGUUAUCCA 1567GfGfccguuaucca AD-397230 asgscau(Uhd)UfuGf 1568usGfscgcAfcAfUfguucAfaAfaugcususu 1569 AAAGCAUUUUGAACAUGUGCGCA 1570AfAfcaugugcgca AD-397231 usgsuga(Uhd)CfuAf 1571usUfscauGfcGfCfucguAfgAfucacascsg 1572 CGUGUGAUCUACGAGCGCAUGAA 1573CfGfagcgcaugaa AD-397233 csasgcg(Ahd)GfaAf 1574usAfsguuAfgUfGfcucuUfcUfcgcugscsa 1575 UGCAGCGAGAAGAGCACUAACUU 1576GfAfgcacuaacua AD-397234 asgscgu(Ghd)UfcAf 1577usAfsaacUfuUfGfgguuGfaCfacgcusgsc 1578 GCAGCGUGUCAACCCAAAGUUUA 1579AfCfccaaaguuua AD-397235 usgsuca(Ahd)CfcCf 1580usGfsaguAfaAfCfuuugGfgUfugacascsg 1581 CGUGUCAACCCAAAGUUUACUCA 1582AfAfaguuuacuca AD-397236 usgsucc(Chd)Aftfa 1583usCfsgccGfuAfAfaagaAfuGfggacascsa 1584 UGUGUCCCAUUCUUUUACGGCGG 1585CfUfuuuacggcga AD-397237 gsusguc(Ahd)AfcCf 1586usAfsguaAfaCfUfuuggGfuUfgacacsgsc 1587 GCGUGUCAACCCAAAGUUUACUC 1588CfAfaaguuuacua AD-397238 asasgau(Chd)CfuGf 1589usGfsggaAfgUfUfuaucAfgGfaucuusgsg 1590 CCAAGAUCCUGAUAAACUUCCCA 1591AfUfaaacuuccca AD-397239 asgsauc(Chd)UfgAf 1592usUfsgggAfaGfUfuuauCfaGfgaucususg 1593 CAAGAUCCUGAUAAACUUCCCAC 1594UfAfaacuucccaa AD-397240 csusuac(Chd)GfuUf 1595usAfsccaAfcUfAfggcaAfcGfguaagsgsa 1596 UCCUUACCGUUGCCUAGUUGGUG 1597GfCfcuaguuggua AD-397241 gsusgug(Uhd)CfcCf 1598usCfsguaAfaAfGfaaugGfgAfcacacsusu 1599 AAGUGUGUCCCAUUCUUUUACGG 1600AfUfucuuuuacga AD-397242 gsusguc(Chd)CfaUf 1601usGfsccgUfaAfAfagaaUfgGfgacacsasc 1602 GUGUGUCCCAUUCUUUUACGGCG 1603UfCfuuuuacggca AD-397243 csasuag(Chd)AfaCf 1604usUfsgacAfaUfCfacggUfuGfcuaugsasc 1605 GUCAUAGCAACCGUGAUUGUCAU 1606CfGfugauugucaa AD-397244 gsasacg(Ghd)AfuAf 1607usUfsuggAfuUfCfucauAfuCfcguucsusg 1608 CAGAACGGAUAUGAGAAUCCAAC 1609UfGfagaauccaaa AD-397245 usgsugu(Chd)CfcAf 1610usCfscguAfaAfAfgaauGfgGfacacascsu 1611 AGUGUGUCCCAUUCUUUUACGGC 1612UfUfcuuuuacgga AD-397246 gscsaac(Chd)GfuGf 1613usGfsugaUfgAfCfaaucAfcGfguugcsusa 1614 UAGCAACCGUGAUUGUCAUCACC 1615AfUfugucaucaca AD-397247 gscsagc(Ghd)AfgAf 1616usGfsuuaGfuGfCfucuuCfuCfgcugcsasu 1617 AUGCAGCGAGAAGAGCACUAACU 1618AfGfagcacuaaca AD-397248 csasgaa(Uhd)UfcGf 1619usUfsgaaUfcAfUfguccGfaAfuucugscsa 1620 UGCAGAAUUCGGACAUGAUUCAG 1621GfAfcaugauucaa AD-397249 uscscug(Ahd)UfaAf 1622usUfscguGfgGfAfaguuUfaUfcaggasusc 1623 GAUCCUGAUAAACUUCCCACGAC 1624AfCfuucccacgaa AD-397250 asgsaac(Ghd)GfaUf 1625usUfsggaUfuCfUfcauaUfcCfguucusgsc 1626 GCAGAACGGAUAUGAGAAUCCAA 1627AfUfgagaauccaa AD-397251 cscsuua(Chd)CfgUf 1628usCfscaaCfuAfGfgcaaCfgGfuaaggsasa 1629 UUCCUUACCGUUGCCUAGUUGGU 1630UfGfccuaguugga AD-397252 asusccu(Ghd)AfuAf 1631usCfsgugGfgAfAfguuuAfuCfaggauscsu 1632 AGAUCCUGAUAAACUUCCCACGA 1633AfAfcuucccacga AD-397253 cscsuga(Uhd)AfaAf 1634usGfsucgUfgGfGfaaguUfuAfucaggsasu 1635 AUCCUGAUAAACUUCCCACGACA 1636CfUfucccacgaca AD-397254 csgsgau(Ghd)GfaUf 1637usGfsucuCfaCfAfaacaUfcCfauccgscsu 1638 AGCGGAUGGAUGUUUGUGAGACC 1639GfUfuugugagaca AD-397255 gsascac(Ghd)GfaAf 1640usAfsugcAfgUfAfcucuUfcCfgugucsasa 1641 UUGACACGGAAGAGUACUGCAUG 1642GfAfguacugcaua AD-397256 gscsagc(Ahd)GfaAf 1643usUfscucAfuAfUfccguUfcUfgcugcsasu 1644 AUGCAGCAGAACGGAUAUGAGAA 1645CfGfgauaugagaa AD-397257 gscsaga(Ahd)CfgGf 1646usGfsauuCfuCfAfuaucCfgUfucugcsusg 1647 CAGCAGAACGGAUAUGAGAAUCC 1648AfUfaugagaauca AD-397258 csasgaa(Chd)GfgAf 1649usGfsgauUfcUfCfauauCfcGfuucugscsu 1650 AGCAGAACGGAUAUGAGAAUCCA 1651UfAfugagaaucca AD-397259 ascscgu(Chd)GfcCf 1652usCfsaugUfcUfCfuuugGfcGfacggusgsu 1653 ACACCGUCGCCAAAGAGACAUGC 1654AfAfagagacauga AD-397260 gsusucu(Ghd)UfgGf 1655usUfsguuGfaGfUfuuacCfaCfagaacsasu 1656 AUGUUCUGUGGUAAACUCAACAU 1657UfAfaacucaacaa AD-397261 gsgsuac(Uhd)UfuGf 1658usUfsucaGfuGfAfcaucAfaAfguaccsasg 1659 CUGGUACUUUGAUGUCACUGAAG 1660AfUfgucacugaaa AD-397262 cscscaa(Ahd)GfuUf 1661usAfsgucUfuGfAfguaaAfcUfuugggsusu 1662 AACCCAAAGUUUACUCAAGACUA 1663UfAfcucaagacua AD-397263 cscsaaa(Ghd)UfuUf 1664usUfsaguCfuUfGfaguaAfaCfuuuggsgsu 1665 ACCCAAAGUUUACUCAAGACUAC 1666AfCfucaagacuaa AD-397264 csasuca(Uhd)GfuGf 1667usAfsgcaUfgUfUfgaacAfcAfugaugsasg 1668 CUCAUCAUGUGUUCAACAUGCUG 1669UfUfcaacaugcua AD-397265 asascau(Ghd)CfuGf 1670usAfscguAfcUfUfcuucAfgCfauguusgsa 1671 UCAACAUGCUGAAGAAGUACGUC 1672AfAfgaaguacgua AD-397266 ususcug(Uhd)GfgUf 1673usAfsuguUfgAfGfuuuaCfcAfcagaascsa 1674 UGUUCUGUGGUAAACUCAACAUG 1675AfAfacucaacaua AD-397267 uscsugu(Ghd)GfuAf 1676usCfsaugUfuGfAfguuuAfcCfacagasasc 1677 GUUCUGUGGUAAACUCAACAUGC 1678AfAfcucaacauga

TABLE 6 APP Unmodified Sequences, Mouse NM_001198823.1 Targeting SEQ SEQDuplex Sense ID Position in Antisense ID Position in NameSequence (5′ to 3′) NO NM_001198823.1 Sequence (5′ to 3′) NONM_001198823.1 AD-397183 CCAUGUUCUGUGGUAAACUCA 1679 253-273UGAGUUUACCACAGAACAUGGCG 1680 251-273 AD-397175 CAUGUUCUGUGGUAAACUCAA1681 254-274 UUGAGUUUACCACAGAACAUGGC 1682 252-274 AD-397177AUGUUCUGUGGUAAACUCAAA 1683 255-275 UUUGAGUUUACCACAGAACAUGG 1684 253-275AD-397176 UGUUCUGUGGUAAACUCAACA 1685 256-276 UGUUGAGUUUACCACAGAACAUG1686 254-276 AD-397260 GUUCUGUGGUAAACUCAACAA 1687 257-277UUGUUGAGUUUACCACAGAACAU 1688 255-277 AD-397266 UUCUGUGGUAAACUCAACAUA1689 258-278 UAUGUUGAGUUUACCACAGAACA 1690 256-278 AD-397267UCUGUGGUAAACUCAACAUGA 1691 259-279 UCAUGUUGAGUUUACCACAGAAC 1692 257-279AD-397178 CUGUGGUAAACUCAACAUGCA 1693 260-280 UGCAUGUUGAGUUUACCACAGAA1694 258-280 AD-397180 UGUGGUAAACUCAACAUGCAA 1695 261-281UUGCAUGUUGAGUUUACCACAGA 1696 259-281 AD-397184 GUGGUAAACUCAACAUGCACA1697 262-282 UGUGCAUGUUGAGUUUACCACAG 1698 260-282 AD-397179GGUAAACUCAACAUGCACAUA 1699 264-284 UAUGUGCAUGUUGAGUUUACCAC 1700 262-284AD-397224 GUACUGCCAAGAGGUCUACCA 1701 362-382 UGGUAGACCUCUUGGCAGUACUG1702 360-382 AD-397216 UACUGCCAAGAGGUCUACCCA 1703 363-383UGGGUAGACCUCUUGGCAGUACU 1704 361-383 AD-397225 ACUGCCAAGAGGUCUACCCUA1705 364-384 UAGGGUAGACCUCUUGGCAGUAC 1706 362-384 AD-397203CUGAACUGCAGAUCACAAACA 1707 382-402 UGUUUGUGAUCUGCAGUUCAGGG 1708 380-402AD-397185 GAACUGCAGAUCACAAACGUA 1709 384-404 UACGUUUGUGAUCUGCAGUUCAG1710 382-404 AD-397195 CACCCACAUCGUGAUUCCUUA 1711 473-493UAAGGAAUCACGAUGUGGGUGUG 1712 471-493 AD-397204 CCACAUCGUGAUUCCUUACCA1713 476-496 UGGUAAGGAAUCACGAUGUGGGU 1714 474-496 AD-397191CACAUCGUGAUUCCUUACCGA 1715 477-497 UCGGUAAGGAAUCACGAUGUGGG 1716 475-497AD-397251 CCUUACCGUUGCCUAGUUGGA 1717 489-509 UCCAACUAGGCAACGGUAAGGAA1718 487-509 AD-397240 CUUACCGUUGCCUAGUUGGUA 1719 490-510UACCAACUAGGCAACGGUAAGGA 1720 488-510 AD-397205 GUGCCCGACAAGUGCAAGUUA1721 534-554 UAACUUGCACUUGUCGGGCACGA 1722 532-554 AD-397254CGGAUGGAUGUUUGUGAGACA 1723 567-587 UGUCUCACAAACAUCCAUCCGCU 1724 565-587AD-397259 ACCGUCGCCAAAGAGACAUGA 1725 603-623 UCAUGUCUCUUUGGCGACGGUGU1726 601-623 AD-397247 GCAGCGAGAAGAGCACUAACA 1727 622-642UGUUAGUGCUCUUCUCGCUGCAU 1728 620-642 AD-397233 CAGCGAGAAGAGCACUAACUA1729 623-643 UAGUUAGUGCUCUUCUCGCUGCA 1730 621-643 AD-397181GAAGAGCACUAACUUGCACGA 1731 629-649 UCGUGCAAGUUAGUGCUCUUCUC 1732 627-649AD-397186 AAGAGCACUAACUUGCACGAA 1733 630-650 UUCGUGCAAGUUAGUGCUCUUCU1734 628-650 AD-397196 AGAGCACUAACUUGCACGACA 1735 631-651UGUCGUGCAAGUUAGUGCUCUUC 1736 629-651 AD-397187 AGCACUAACUUGCACGACUAA1737 633-653 UUAGUCGUGCAAGUUAGUGCUCU 1738 631-653 AD-397188GCACUAACUUGCACGACUAUA 1739 634-654 UAUAGUCGUGCAAGUUAGUGCUC 1740 632-654AD-397197 CACUAACUUGCACGACUAUGA 1741 635-655 UCAUAGUCGUGCAAGUUAGUGCU1742 633-655 AD-397226 ACUAACUUGCACGACUAUGGA 1743 636-656UCCAUAGUCGUGCAAGUUAGUGC 1744 634-656 AD-397212 UUGCACGACUAUGGCAUGCUA1745 642-662 UAGCAUGCCAUAGUCGUGCAAGU 1746 640-662 AD-397182CCGCUGGUACUUUGAUGUCAA 1747 1064-1084 UUGACAUCAAAGUACCAGCGGGA 17481062-1084 AD-397261 GGUACUUUGAUGUCACUGAAA 1749 1069-1089UUUCAGUGACAUCAAAGUACCAG 1750 1067-1089 AD-397241 GUGUGUCCCAUUCUUUUACGA1751 1094-1114 UCGUAAAAGAAUGGGACACACUU 1752 1092-1114 AD-397245UGUGUCCCAUUCUUUUACGGA 1753 1095-1115 UCCGUAAAAGAAUGGGACACACU 17541093-1115 AD-397242 GUGUCCCAUUCUUUUACGGCA 1755 1096-1116UGCCGUAAAAGAAUGGGACACAC 1756 1094-1116 AD-397236 UGUCCCAUUCUUUUACGGCGA1757 1097-1117 UCGCCGUAAAAGAAUGGGACACA 1758 1095-1117 AD-397227GUCCCAUUCUUUUACGGCGGA 1759 1098-1118 UCCGCCGUAAAAGAAUGGGACAC 17601096-1118 AD-397255 GACACGGAAGAGUACUGCAUA 1761 1143-1163UAUGCAGUACUCUUCCGUGUCAA 1762 1141-1163 AD-397234 AGCGUGUCAACCCAAAGUUUA1763 1176-1196 UAAACUUUGGGUUGACACGCUGC 1764 1174-1196 AD-397237GUGUCAACCCAAAGUUUACUA 1765 1179-1199 UAGUAAACUUUGGGUUGACACGC 17661177-1199 AD-397235 UGUCAACCCAAAGUUUACUCA 1767 1180-1200UGAGUAAACUUUGGGUUGACACG 1768 1178-1200 AD-397262 CCCAAAGUUUACUCAAGACUA1769 1186-1206 UAGUCUUGAGUAAACUUUGGGUU 1770 1184-1206 AD-397263CCAAAGUUUACUCAAGACUAA 1771 1187-1207 UUAGUCUUGAGUAAACUUUGGGU 17721185-1207 AD-397189 AAAGUUUACUCAAGACUACCA 1773 1189-1209UGGUAGUCUUGAGUAAACUUUGG 1774 1187-1209 AD-397198 CUCAAGACUACCAGUGAACCA1775 1197-1217 UGGUUCACUGGUAGUCUUGAGUA 1776 1195-1217 AD-397206GACUACCAGUGAACCUCUUCA 1777 1202-1222 UGAAGAGGUUCACUGGUAGUCUU 17781200-1222 AD-397238 AAGAUCCUGAUAAACUUCCCA 1779 1225-1245UGGGAAGUUUAUCAGGAUCUUGG 1780 1223-1245 AD-397239 AGAUCCUGAUAAACUUCCCAA1781 1226-1246 UUGGGAAGUUUAUCAGGAUCUUG 1782 1224-1246 AD-397252AUCCUGAUAAACUUCCCACGA 1783 1228-1248 UCGUGGGAAGUUUAUCAGGAUCU 17841226-1248 AD-397249 UCCUGAUAAACUUCCCACGAA 1785 1229-1249UUCGUGGGAAGUUUAUCAGGAUC 1786 1227-1249 AD-397253 CCUGAUAAACUUCCCACGACA1787 1230-1250 UGUCGUGGGAAGUUUAUCAGGAU 1788 1228-1250 AD-397217CACCGAGAGAGAAUGUCCCAA 1789 1353-1373 UUGGGACAUUCUCUCUCGGUGCU 17901351-1373 AD-397213 UCCCAGGUCAUGAGAGAAUGA 1791 1368-1388UCAUUCUCUCAUGACCUGGGACA 1792 1366-1388 AD-397228 AAGCUGACAAGAAGGCCGUUA1793 1423-1443 UAACGGCCUUCUUGUCAGCUUUG 1794 1421-1443 AD-397229UGACAAGAAGGCCGUUAUCCA 1795 1427-1447 UGGAUAACGGCCUUCUUGUCAGC 17961425-1447 AD-397208 GGCCCUCGAGAAUUACAUCAA 1797 1562-1582UUGAUGUAAUUCUCGAGGGCCAG 1798 1560-1582 AD-397218 CAAGGCCUCAUCAUGUGUUCA1799 1603-1623 UGAACACAUGAUGAGGCCUUGGG 1800 1601-1623 AD-397264CAUCAUGUGUUCAACAUGCUA 1801 1611-1631 UAGCAUGUUGAACACAUGAUGAG 18021609-1631 AD-397265 AACAUGCUGAAGAAGUACGUA 1803 1623-1643UACGUACUUCUUCAGCAUGUUGA 1804 1621-1643 AD-397209 CAUGCUGAAGAAGUACGUCCA1805 1625-1645 UGGACGUACUUCUUCAGCAUGUU 1806 1623-1645 AD-397192AUGCUGAAGAAGUACGUCCGA 1807 1626-1646 UCGGACGUACUUCUUCAGCAUGU 18081624-1646 AD-397210 UGCUGAAGAAGUACGUCCGUA 1809 1627-1647UACGGACGUACUUCUUCAGCAUG 1810 1625-1647 AD-397219 GCUGAAGAAGUACGUCCGUGA1811 1628-1648 UCACGGACGUACUUCUUCAGCAU 1812 1626-1648 AD-397214CUGAAGAAGUACGUCCGUGCA 1813 1629-1649 UGCACGGACGUACUUCUUCAGCA 18141627-1649 AD-397199 AGCACACCCUAAAGCAUUUUA 1815 1666-1686UAAAAUGCUUUAGGGUGUGCUGU 1816 1664-1686 AD-397220 AAGCAUUUUGAACAUGUGCGA1817 1677-1697 UCGCACAUGUUCAAAAUGCUUUA 1818 1675-1697 AD-397230AGCAUUUUGAACAUGUGCGCA 1819 1678-1698 UGCGCACAUGUUCAAAAUGCUUU 18201676-1698 AD-397221 CACCUCCGUGUGAUCUACGAA 1821 1746-1766UUCGUAGAUCACACGGAGGUGUG 1822 1744-1766 AD-397215 CGUGUGAUCUACGAGCGCAUA1823 1752-1772 UAUGCGCUCGUAGAUCACACGGA 1824 1750-1772 AD-397231UGUGAUCUACGAGCGCAUGAA 1825 1754-1774 UUCAUGCGCUCGUAGAUCACACG 18261752-1774 AD-397193 GAGCGCAUGAACCAGUCUCUA 1827 1764-1784UAGAGACUGGUUCAUGCGCUCGU 1828 1762-1784 AD-397190 CGCAUGAACCAGUCUCUGUCA1829 1767-1787 UGACAGAGACUGGUUCAUGCGCU 1830 1765-1787 AD-397222GAAGGAGCAGAACUACUCCGA 1831 1850-1870 UCGGAGUAGUUCUGCUCCUUCUG 18321848-1870 AD-397200 AAGGAGCAGAACUACUCCGAA 1833 1851-1871UUCGGAGUAGUUCUGCUCCUUCU 1834 1849-1871 AD-397201 GGAGCAGAACUACUCCGACGA1835 1853-1873 UCGUCGGAGUAGUUCUGCUCCUU 1836 1851-1873 AD-397194GAGCAGAACUACUCCGACGAA 1837 1854-1874 UUCGUCGGAGUAGUUCUGCUCCU 18381852-1874 AD-397248 CAGAAUUCGGACAUGAUUCAA 1839 2167-2187UUGAAUCAUGUCCGAAUUCUGCA 1840 2165-2187 AD-397207 GUCCGCCAUCAAAAACUGGUA1841 2196-2216 UACCAGUUUUUGAUGGCGGACUU 1842 2194-2216 AD-397211UCCGCCAUCAAAAACUGGUGA 1843 2197-2217 UCACCAGUUUUUGAUGGCGGACU 18442195-2217 AD-397243 CAUAGCAACCGUGAUUGUCAA 1845 2282-2302UUGACAAUCACGGUUGCUAUGAC 1846 2280-2302 AD-397246 GCAACCGUGAUUGUCAUCACA1847 2286-2306 UGUGAUGACAAUCACGGUUGCUA 1848 2284-2306 AD-397223GAAGAAACAGUACACAUCCAA 1849 2321-2341 UUGGAUGUGUACUGUUUCUUCUU 18502319-2341 AD-397202 GAAACAGUACACAUCCAUCCA 1851 2324-2344UGGAUGGAUGUGUACUGUUUCUU 1852 2322-2344 AD-397256 GCAGCAGAACGGAUAUGAGAA1853 2405-2425 UUCUCAUAUCCGUUCUGCUGCAU 1854 2403-2425 AD-397257GCAGAACGGAUAUGAGAAUCA 1855 2408-2428 UGAUUCUCAUAUCCGUUCUGCUG 18562406-2428 AD-397258 CAGAACGGAUAUGAGAAUCCA 1857 2409-2429UGGAUUCUCAUAUCCGUUCUGCU 1858 2407-2429 AD-397250 AGAACGGAUAUGAGAAUCCAA1859 2410-2430 UUGGAUUCUCAUAUCCGUUCUGC 1860 2408-2430 AD-397244GAACGGAUAUGAGAAUCCAAA 1861 2411-2431 UUUGGAUUCUCAUAUCCGUUCUG 18622409-2431

TABLE 7 APP Single Dose Screen in Primary Mouse Hepatocytes and Neuro2ACell Line Data are expressed as percent message remaining relative toAD-1955 non-targeting control. Primary Mouse Hepatocytes Neuro2A CellLine 10 nM 10 nM 0.1 nM 0.1 nM 10 nM 10 nM 0.1 nM 0.1 nM Duplex Name AvgSD Avg SD Avg SD Avg SD AD-397183 4.2 1.4 37.3 24.3 7.94 2.86 52.66 5.87AD-397175 1.6 0.7 4.7 1.3 0.75 0.32 29.72 6.47 AD-397177 1.3 1.1 3.9 2.60.4 0.13 18.06 3.73 AD-397176 1.5 0.5 35.1 11.3 4.7 1.45 69.36 7.89AD-397260 11.2 1.5 73.4 23.1 20.53 3.62 81.33 2.21 AD-397266 2.8 2 65.14.5 4.35 0.58 73.16 8.45 AD-397267 0.8 0.3 23.6 4.2 1.18 0.28 37.78 3.45AD-397178 5.1 4.1 33.3 6.1 1.8 0.38 54.61 3.11 AD-397180 1.3 0.4 28 13.90.47 0.06 37.8 3.96 AD-397184 15.7 8.9 67.8 13.5 8.86 2.55 87.82 5.6AD-397179 5.7 1.6 45.1 26 3.12 0.86 57.24 5.19 AD-397224 52.9 18.5 63.810.6 17.15 2.47 67.99 7.6 AD-397216 25.6 17.9 104.2 21.6 34.91 7.4498.89 4.08 AD-397225 45.1 21.9 60.8 13.7 9.72 5.52 63.44 7.19 AD-3972033.3 1.6 71.9 8.2 5.1 0.98 75.87 3.29 AD-397185 4.9 2.1 40.3 8.1 2.7 0.3561.49 8.12 AD-397195 2.5 1.3 49.8 21.8 1.64 0.08 63.95 5.83 AD-3972048.3 2 68 10.7 4.37 0.89 50.83 7.41 AD-397191 1.5 0.5 39.9 14.8 1.5 1.0655.07 10.78 AD-397251 7.8 1.7 91.7 5.7 3.86 2.5 84.36 6.5 AD-397240 4.21.9 61.9 6.8 2.48 0.7 62.39 1.48 AD-397205 13.5 10.5 86 11.4 13.06 7.6176.77 2.64 AD-397254 1.9 1.1 27.6 24.3 3.77 2.77 57.26 14.42 AD-3972593.5 0.7 79 22.8 9.43 1.12 82.49 3.19 AD-397247 5.5 1 90.4 16.9 10.952.85 94.95 4.55 AD-397233 6.7 6.2 84.4 10.3 3.4 1.14 76.36 4.66AD-397181 4.7 0.9 60.5 25.2 6.28 2.17 62.62 3.59 AD-397186 53 17 82 14.742.07 9.63 95.63 6.67 AD-397196 1.9 0.4 40.9 11.3 4.66 4.19 56.2 1.82AD-397187 28.4 11.2 77.5 13.3 25.64 8.56 86.64 5.99 AD-397188 65.1 15.976.2 20 43.32 13.51 84.69 5.63 AD-397197 2 1 41.9 10.7 2.11 0.41 55.632.15 AD-397226 10.3 4.3 30 5 0.69 0.43 47.42 5.33 AD-397212 1.8 0.1 65.49.3 1.94 0.48 63 29.9 AD-397182 2.1 0.6 11.3 5.3 12.2 3.42 35.13 6.78AD-397261 2.3 0.6 32.6 10 29.93 2.71 48.28 24.73 AD-397241 23 3.5 102.713.3 41.16 4.58 92.7 5.11 AD-397245 60.9 8.6 60.9 14.3 55.71 4.45 68.276.83 AD-397242 5.6 1.1 90.5 16.2 30.83 2.94 85.43 4.05 AD-397236 16.96.2 71.9 5.7 32.58 2.93 67.13 3.06 AD-397227 48.7 29.8 50.5 19.4 19.559.28 59.59 3.24 AD-397255 6.1 0.8 73.8 33 24.01 5 86.3 9.24 AD-397234100.3 39.9 93.7 7.8 51.88 13.54 80.77 2.1 AD-397237 36.2 28.6 49.5 1432.83 17.93 51.76 10.71 AD-397235 58 20.9 76.2 8 41.15 19.69 73.72 6AD-397262 22.1 6.9 51.8 16.2 61.74 5.34 65.6 14.12 AD-397263 19.9 8 57.96.1 59.09 7.38 82.09 11.31 AD-397189 17 5.1 56.2 9.5 49.48 18.93 73.895.4 AD-397198 19.8 2.4 38.8 9.1 50.52 28.37 62.16 9.56 AD-397206 18.81.7 41 12.6 62.65 21.77 61.59 8.42 AD-397238 16.3 2 61.5 27.8 71.66 9.386.52 7.97 AD-397239 34.6 11.4 101 22.8 74.11 7.37 91.24 4.34 AD-39725223.1 7.5 93.8 3.1 55.54 4.89 75.74 5.31 AD-397249 35.6 4 104.9 10.970.19 3.96 97.86 6.43 AD-397253 29.6 5.5 44.6 19.2 66.41 8.65 66.4 6.46AD-397217 11.5 6.3 102.4 20.9 18.85 3.87 98.69 3.04 AD-397213 7.3 1.979.4 21.9 10.91 2.81 87.03 4.86 AD-397228 68.7 66.7 43.2 9.3 23.79 8.4553.36 3.55 AD-397229 3.9 0.3 15.8 9.4 1.67 1.35 31.6 5.21 AD-397208 18.23.9 96.2 27.2 37.55 9.28 97.91 5.09 AD-397218 35 14.6 106 20.7 30.887.34 101.82 3.13 AD-397264 4.2 2.2 98 12.9 19.97 2.06 104.79 4.61AD-397265 3 2.3 81.2 7.8 5.98 4.03 84.1 8.97 AD-397209 10.9 9.3 90.522.2 17.18 3.16 81.66 5.17 AD-397192 4.7 1.8 80.6 13 6.51 1.99 95.044.22 AD-397210 22.6 6.4 83.6 24.7 6.55 1.38 82.6 3.83 AD-397219 10.2 3.6101.8 21.8 16.76 3.62 87.34 4.87 AD-397214 5.8 0.9 34.4 14.3 12.78 5.2454.95 18.66 AD-397199 62.2 14.3 63.4 35 87.69 22.23 85.84 4.93 AD-3972205.2 0.5 99.2 18.2 5.91 1.12 91.13 2.97 AD-397230 6.3 3.9 61.5 23.1 5.513.99 77.38 3.26 AD-397221 10.5 3.4 111.2 42.5 24.53 4.87 93.86 3.22AD-397215 14.3 2.9 80.7 40 44.04 14.01 91.83 10.03 AD-397231 17.1 3.2108.7 19.6 21.54 1.56 79.31 4.22 AD-397193 3.3 0.3 93.1 21.6 12.76 1.9793.03 6.46 AD-397190 2.7 0.5 27.8 13.5 3.63 2.79 45.56 7.21 AD-39722262.9 9.1 57.2 17 25.04 11.48 80.41 4.04 AD-397200 8.6 8.2 89.6 18.6 9.631.79 88.31 6.27 AD-397201 85.2 40.7 106 17.5 41.76 9.95 105.41 3.36AD-397194 35.4 12.2 92 8.3 51.26 11.38 107.07 3.23 AD-397248 7.8 1.197.5 17.7 17.64 1.67 103.37 4.94 AD-397207 6.9 4 59.5 39.1 6.28 2.6582.18 8.76 AD-397211 18.2 8.6 101.1 20.6 14.71 4.06 96.99 2.56 AD-3972432.2 1.5 63.1 11.2 0.6 0.32 55.57 2.17 AD-397246 1.5 0.6 46.6 22.5 0.860.64 63.09 3.39 AD-397223 46.8 15.8 63.3 17.2 9.73 2.48 73.44 2.51AD-397202 32.5 7.6 103.4 25.9 20.68 4.37 95.57 5.11 AD-397256 2.1 0.771.4 8 1.77 1.21 79.93 1.89 AD-397257 2.4 0.7 76.1 23.3 5.45 2.7 84.437.45 AD-397258 0.9 0.2 45.4 8.3 0.63 0.4 55.81 5.17 AD-397250 0.8 0.154.9 11.3 0.52 0.23 46.87 3.19 AD-397244 2.2 1.2 74.2 12 1.87 1.87 67.153.5As noted for Table 4 above, it is expressly contemplated that any RNAiagents possessing target sequences that reside fully within thefollowing windows of NM_001198823.1 positions are likely to exhibitrobust APP inhibitory effect: APP NM_001198823.1 positions 251-284; APPNM_001198823.1 positions 362-404; APP NM_001198823.1 positions 471-510;APP NM_001198823.1 positions 532-587; APP NM_001198823.1 positions601-649; APP NM_001198823.1 positions 633-662; APP NM_001198823.1positions 1351-1388; APP NM_001198823.1 positions 1609-1649; APPNM_001198823.1 positions 1675-1698; APP NM_001198823.1 positions1752-1787; APP NM_001198823.1 positions 2165-2217; APP NM_001198823.1positions 2280-2344; and APP NM_001198823.1 positions 2403-2431.

Example 2. In Vivo Evaluation of RNAi Agents

Selected APP-targeting RNAi agents were evaluated for in vivo efficacyin respective proof of concept and lead identification screens for humanAPP knockdown in AAV mice. The selected RNAi agents for such studiesincluded AD-392911, AD-392912, AD-392911, AD-392912, AD-392913,AD-392843, AD-392844, AD-392824, AD-392704, AD-392790, AD-392703,AD-392866, AD-392927, AD-392916, AD-392714 and AD-392926, havingsequences as recited in Table 2A above, corresponding unmodifiedsequences as shown in Table 3 above, and as graphically depicted in FIG.1A and FIG. 1B, with each RNAi agent tested in the instant Examplefurther presenting a triantennary GalNAc moiety attached at the 3′residue of the sense strand, for purpose of aiding liver targeting ofsuch RNAi agents when administered subcutaneously to mice (forintrathecal administration, agents lacking a conjugated GalNAc moietyare expressly contemplated).

In such studies, an AAV vector harboring Homo sapiens APP wasintravenously injected to 6-8 week old C57BL/6 female mice, and at 14days post-AAV administration, a selected RNAi agent or a control agentwere subcutaneously injected at 3 mg/kg to mice (n=3 per group), withmice sacrificed and livers assessed for APP mRNA levels at 14 dayspost-subcutaneous injection of RNAi agent or control. Significant levelsof in vivo human APP mRNA knockdown in liver were observed for all RNAiagents tested, as compared to PBS and Naïve (AAV only) controls, withparticularly robust levels of knockdown observed, e.g., for AD-392911,AD-392912, AD-392911, AD-392912, AD-392913, AD-392843, AD-392844,AD-392824, AD-392866, AD-392927, AD-392916, AD-392714 and AD-392926(FIG. 2A and FIG. 2B). Results used to generate FIG. 2A and FIG. 2B aretabulated in below Table 8.

TABLE 8 hsAPP In Vivo Knockdown Screen Results (3 mg/kg, day 14, liver)% message Treatment remaining stdev PBS 100.00 15.77 naïve (AAV-only)104.17 1.89 AD-392911 53.75 8.76 AD-392912 46.47 14.18 AD-392913 42.347.95 AD-392843 27.25 0.46 AD-392844 44.25 9.04 AD-392824 42.64 0.87AD-392704 72.99 8.76 AD-392790 72.71 11.66 AD-392703 69.60 4.70AD-392866 35.94 23.08 AD-392927 38.91 10.60 AD-392916 43.27 7.17AD-392714 58.08 9.55 AD-392926 50.26 10.29

Example 3: Identification of Potent Human APP siRNAs Against HereditaryCerebral Amyloid Angiopathy (hCAA)

Hereditary cerebral amyloid angiopathy (hCAA) is driven by autosomaldominant mutations in the gene encoding Amyloid Precursor Protein (APP)(Van Etten et al. 2016 Neurology). In the disease, neuron-derived betaamyloid is deposited in vasculature causing significant structuralalterations and a distinctive double barreling of vessels. hCAA appearsto be a relatively pure angiopathy with minimal presence of parenchymalplaques or tau tangles (Natte et al. 2012 Annals of Neurology).Ultimately, increased deposition of amyloid beta leads tomicrohemorrhages, dementia and stroke. hCAA is a rapidly progressingdisease with life expectancy of 7-10 years following symptom onset(Charidimou A et al. J Neurol Neurosurg Psychiatry 2012; 83: 124-137).As noted herein, there are currently no disease-modifying therapiesavailable. In the instant disclosure, combining stable siRNA designswith alternative conjugation strategies provided potent, long-lastingsilencing across the CNS following a single intrathecal administrationwith 95% target knockdown observed out to three months.

Be(2)C Cell Screening and In Vivo Liver Based Screens

To identify potent hAPP siRNAs, siRNAs were first screened in vitro inBe(2)C cells. As shown in FIG. 3A and FIG. 3B, over 300 siRNAs weretransfected into Be(2)C cells at concentrations of 10 nM (FIG. 3B) and0.1 nM (data not shown) and the percent remaining mRNA was assayed byqPCR. In vivo liver based AAV-hAPP screening was then performed in micein order to identify compounds capable of knocking down human APP.GalNAc APP siRNAs designed against either hAPP ORF or hAPP 3′ UTR wereadministered subcutaneously at 3 mg/kg (as shown in FIGS. 2A and 2B,respectively). A selected subset of compounds was then converted to CNSconjugates and used in both non-human primate lead finding studies andin rodent models of disease using intrathecal (IT) administration. Asnoted above, particularly robust levels of knockdown were observed for,e.g., AD-392911, AD-392912, AD-392911, AD-392912, AD-392913, AD-392843,AD-392844, AD-392824, AD-392866, AD-392927, AD-392916, AD-392714 andAD-392926 (FIG. 2A and FIG. 2B).

APP siRNA transfected at 10 nM, 1 nM, and 0.1 nM into Be(2)C neuronalcells was evaluated for knockdown of APP mRNA, as well as soluble AAPα/β levels, at both 24 and 48 hours after transfection (see e.g., FIG.4A, FIG. 4B, and FIG. 4C). A concentration dependent knockdown of APPmRNA was observed for both example siRNAs of interest (e.g., siRNA 1 andsiRNA 2 shown in FIGS. 4A-4C). Further, a reduction of cellular APPcorresponded to an up to 99% knockdown of soluble AAP α/β in Be(2)Cneuronal cell within 48 hours.

Example 4: Intrathecal (IT) Dosing Delivered APP siRNA Throughout theSpinal Cord and Brain of Non-Human Primates

Non-Human Primate Studies

Dose Formulation and Preparation

Test Oligonucleotides and Vehicle Information

Test Oligonucleotides: AD-454972

-   -   AD-454973    -   AD-454842    -   AD-454843    -   AD-454844

The current state of scientific knowledge and the applicable guidelinescited previously in this protocol do not provide acceptablealternatives, in vitro or otherwise, to the use of live animals toaccomplish the purpose of this study. The development of knowledgenecessary for the improvement of the health and well-being of humans aswell as other animals requires in vivo experimentation with a widevariety of animal species. Whole animals are essential in research andtesting because they best reflect the dynamic interactions between thevarious cells, tissues, and organs comprising the human body. The beagleis the usual non-rodent model used for evaluating the toxicity ofvarious test articles and for which there is a large historicaldatabase. However, the monkey is also an animal model used to evaluatetoxicity. The monkey was selected specifically for use in this studybecause it is the pharmacologically relevant species. The siRNA in thetest oligonucleotides is directed against the amyloid precursor protein(APP) mRNA target sequence in monkeys and humans.

STUDY DESIGN Number Dose Level Dose Dose of (mg/animal volumeConcentration Animals Necropsy Necropsy Group Treatment fixed dose) (mL)(mg/mL) (total) (Day 29) (Day 85) 1 AD-454972 72 2.4 30 5 3 2 2AD-454973 72 2.4 30 5 3 2 3 AD-454842 72 2.4 30 5 3 2 4 AD-454843 72 2.430 5 3 2 5 AD-454844 72 2.4 30 5 3 2  6* No Treatment 0 0 0 2 2 0 *Usedfor tissues collection to provide normal tissue, CSF, and plasma levelsof APP in cynomolgus primates. Animals from Groups 1 to 5 withunsuccessful intrathecal cannulation may have been exchanged for thoseassigned Group 6 animals if no oligonucleotide was given. Animals werenecropsied at or before Day 29.The sequence and structure of the oligonucleotides used in theaforementioned non-human primate studies are described in greater detailin Table 9, below.

TABLE 9 Strand SEQ SEQ Agent (Target) Modified Sequence ID NO:Unmodified Sequence ID NO: AD-454972 Senseusasuga(Ahd)GfuUfCfAfucaucaaasasa 1863 UAUGAAGUUCAUCAUCAAAAA 1864 (APP)Antis VPusUfsuuug(Agn)ugaugaAfcUfucauasusc 1865 UUUUUGAUGAUGAACUUCAUAUC1866 (APP) AD-454973 Sense gsgscua(Chd)GfaAfAfAfuccaaccusasa 1867GGCUACGAAAAUCCAACCUAA 1868 (APP) AntisVPusUfsaggu(Tgn)ggauuuUfcGfuagccsgsu 1869 UUAGGUTGGAUUUUCGUAGCCGU 1870(APP) AD-454842 Sense ususugu(Ghd)UfaCfUfGfuaaagaaususa 1871UUUGUGUACUGUAAAGAAUUA 1872 (APP) AntisVPusAfsauuc(Tgn)uuacagUfaCfacaaaasasc 1873 UAAUUCTUUACAGUACACAAAAC 1874(APP) AD-454843 Sense usasgug(Chd)AfuGfAfAfuagauucuscsa 1875UAGUGCAUGAAUAGAUUCUCA 1876 (APP) AntisVPusGfsagaa(Tgn)cuauucAfuGfcacuasgsu 1877 UGAGAATCUAUUCAUGCACUAGU 1878(APP) AD-454844 Sense asasaau(Chd)CfaAfCfCfuacaaguuscsa 1879AAAAUCCAACCUACAAGUUCA 1880 (APP) AntisVPusGfsaacu(Tgn)guagguUfgGfauuuuscsg 1881 UGAACUTGUAGGUUGGAUUUUCG 1882(APP) Table 9 key: U = uridine-3′-phosphate, u= 2′-O-methyluridine-3′-phosphate, us = 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf = 2′-fluoroguanosine-3′-phosphate, Uf = fluorouridine-3′-phosphate, Cf= 2′-fluorocytidine-3′-phosphate, Af = 2′-fluoroadenosine-3′-phosphate,cs = 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn)= Adenosine-glycol nucleic acid (GNA), gs= 2′-O-methylguanosine-3′-phosphorothioate, (Chd) = 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) =  2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

The following are non-limiting examples of knockdown of CSF biomarkerand tissue mRNA via intrathecal (IT) injection of 72 mg drug to the CNStissues of cynomolgus monkeys. A single IT injection, via percutaneousneedle stick, of 72 mg of an APP siRNA of interest was administered incynomolgus monkeys between L2/L3 or L4/L5 in the lumbar cistern (seeMethods and Materials below). As shown in FIG. 5A, 5 compounds wereassessed, and 5 animals were used for each experiment. Tissues collectedwere spinal cord (lumbar, thoracic, and cervical) and brain (prefrontalcortex, temporal cortex, cerebellum, brain stem, hippocampus, andstriatum). Additionally, collected fluids included both cerebrospinalfluid (CSF) and plasma. Drug levels and mRNA knockdown were assessed atday 29 post dose. As shown in FIG. 5B, APP cc/13, as well as amyloidbeta 38, 40, and 42, served as circulating target engagement biomarkersin the CSF and were assessed at days 8, 15, and 29 post-dose. Knockdownin the tissue corresponded to silencing of target engagement biomarkersin the CSF as early as 7 days post dose. As shown in FIG. 5C, IT dosingresulted in sufficient siRNA delivery throughout the spine and brain toresult in APP mRNA knockdown at the tissue level. Tested drug levelswere assessed by mass spectrometry and are shown in FIG. 5D. In summary,FIGS. 5A-5D show the correlation between CSF biomarker levels, mRNAknockdown, and CNS drug delivery of the APP siRNA AD-454972. Thus, itwas notably discovered that CSF biomarker levels and tissue mRNAknockdown exhibited a rapid, robust, and sustained decrease in responseto siRNA conjugate drug levels in the CNS. FIG. 6 demonstrates thatthere is a sustained pharmacodynamic effect observed in the CSF fortarget engagement biomarkers 2-3 months post dose AD-454972.

FIG. 7A shows the results of AD-454842 on sAPP α/β in the CSF, whileFIG. 7B shows tested drug levels of AD-454842 in tissue assessed by massspectrometry. In summary, FIGS. 7A-7B show that CSF biomarker levelscorrelate with drug levels in the CNS for AD-454842, and result in asignificant lowering of sAPP in animals with higher tissue drug levels.

FIG. 8A shows the results of AD-454843 on sAPP α/β and amyloid betaspecies, respectively, in CSF. As shown in FIG. 8B, IT dosing resultedin sufficient siRNA delivery throughout the spine, hippocampus, andcortex regions to result in APP mRNA knockdown at the tissue level.Tested drug levels were assessed by mass spectrometry and are shown inFIG. 8C. Accordingly, FIGS. 8A-8C show a clear correlation between CSFbiomarker levels, mRNA knockdown, and CNS drug delivery of AD-454843.

FIGS. 9A-9B demonstrate a sustained pharmacodynamic effect observed inthe CSF for target engagement biomarkers 2-3 months post-dose forAD-454843. Up to 80% knockdown was observed at the mRNA level in CNStissue at day 85 post dose in cynomolgus monkeys.

FIGS. 10A-10C show the correlation between CSF biomarker levels, mRNAknockdown, and CNS drug delivery for AD-454844. Tested drug levels wereassessed by mass spectrometry and are shown in FIG. 10C.

FIGS. 11A-11C show that optimal delivery of the APP lead siRNAdemonstrates robust activity. For example, the results of high levels ofthe drug on mRNA knockdown and silencing of target engagement biomarkersshows that high μg/g drug levels in tissue correlated with a 75-90%knockdown in CNS tissues such as the cortex and spine. Surprisingly,optimal delivery also showed significant knockdown in the striatum.

FIG. 12A shows the average of 5 duplexes; collectively, IT dosingresulted in sufficient siRNA delivery such that APP mRNA was knockeddown by 60-75% at the tissue level at day 29. Further, as shown in FIG.12B, soluble APP α/β, as well as amyloid beta 38, 40 and 42, werelowered by 75% in the CSF at day 29.

APP mRNA Knockdown in Non-Human Primate Striatum at Day 29 Post Dose

A single intrathecal (IT) injection, via percutaneous needle stick, of72 mg of the APP siRNA of interest was administered in cynomolgusmonkeys between L2/L3 or L4/L5 in the lumbar cistern. In the instantdisclosure, the notable discovery was made that siRNA conjugate compounddelivery resulted in APP mRNA knockdown within the striatum. Thefollowing siRNAs were observed to knockdown APP mRNA in non-humanprimate striatum at day 29 post dose: AD-454972, AD-454973, AD-454842,AD-454843, and AD-454844 (as shown in FIGS. 13A-13E).

Materials and Methods

Soluble APP Alpha/Soluble APP Beta

CSF levels of sAPPα and sAPPβ were determined utilizing a sandwichimmunoassay MSD® 96-well MULTI-SPOT sAPPα/sAPPβ assay (Catalog no.K15120E; Meso Scale Discovery, Rockville, Md., USA) according to themanufacturer's protocol with some modifications. The standards, blanks,and non-human primate CSF samples (8× dilution) were prepared with the1% Blocker-A/TBST (provided in the kit). Pre-coated plate (provided inthe kit) was blocked with 150 μL/well of 3% Blocker A/TBST solution atroom temperature for 1 hour with shaking. After three washes with1×TBST, 25 μL/well of prepared standard, blanks, and CSF samples wereadded to the plate in two replicates and incubated for 1 hour at roomtemperature with shaking. Following subsequent plate washes, 50 μL/wellof detection antibody prepared in 1% Blocker A/TBST (50× dilution) wasadded and incubated at room temperature for 1 hour with shaking. Afterplate washes, 1× Read Buffer T was added to the plate and incubated for10 minutes at room temperature (without shaking) before imaging andanalyzing in MSD QuickPlex Imager.

Raw data were analyzed using SoftMax Pro, version 7.1 (MolecularDevices). A 5-parameter, logistic curve fitting with 1/Y² weighingfunction was used to model the individual calibration curves andcalculate the concentration of analytes in the samples.

Beta Amyloid Panel (Aβ40, Aβ38, Aβ42)

CSF levels of Beta-amyloid (Aβ40, Aβ38, Aβ42) were determined utilizinga sandwich immunoassay multiplex kit MSD® 96-well MULTI-SPOT AB PeptidePanel 1 V-Plex (Catalog No. K15200E, Meso Scale Discovery, Rockville,Md., USA) according to the manufacturer's protocol with somemodifications. The standards, blanks, and non-human primate CSF (8×dilution) were prepared with Diluent 35 (provided in the kit). Detectionantibody (supplied at 50×) was prepared at a working concentration of 1×in Diluent 100 (provided in the kit) combined with 30 μL of A1340Blocker. Pre-coated plate (provided in the kit) was blocked with 150μL/well with Diluent 35 for 1 hour at room temperature with shaking.After three washes with 1×PBST, 25 μl/well of prepared detectionantibody solution was added to the plate. Following with the addition of25 μL/well of prepared standards, blanks, and samples in two replicates,plate was incubated at room temperature for 2 hours with shaking.Following subsequent plate washes, 150 μL/well of 2× Read buffer T wasadded and plate was imaged and analyzed in the MSD QuickPlex Imagerimmediately.

Raw data were analyzed using SoftMax Pro, version 7.1 (MolecularDevices, San Jose, Calif., USA). A 4-parameter, logistic curve fittingwith 1/Y² weighing function was used to model the individual calibrationcurves and calculate the concentration of analytes in the samples.

Mass Spec Method

Drug concentrations in plasma, CSF and CNS tissue samples werequantitated using a qualified LC-MS/MS method. Briefly, tissue sampleswere homogenized in lysis buffer, then the oligonucleotides wereextracted from plasma, CSF or tissue lysate by solid phase extractionand analyzed using ion-pairing reverse phase liquid chromatographycoupled with mass spectrometry under negative ionization mode. Theconcentration of the full-length antisense strand of the dosed duplexwas measured. The drug concentrations were reported as theantisense-based duplex concentrations. The calibration range is 10-5000ng/mL for plasma and CSF samples, and 100-50000 ng/g for CNS tissuesamples. Concentrations that were calculated below the LLOQ are reportedas <LLOQ. An analog duplex with different molecular weight was used asinternal standard.

mRNA Knockdown by qPCR Method

Total RNA was isolated from rat brain and spinal cord tissue samplesusing the miRNeasy Mini Kit from (Qiagen, Catalog No. 217004) accordingto the manufacturer's instructions. Following isolation, RNA was reversetranscribed using SuperScript™ IV VILO™ Reverse Transcriptase (ThermoFisher Scientific). Quantitative PCR analysis was performed using aViiA7 Real-Time PCR System from Thermo Fisher Scientific of WalthamMass. 02451 (Catalog No. 4453537) with Taqman Fast Universal PCR MasterMix (Applied Biosystems Catalog No. 4352042), pre-validated amyloid betaprecursor protein (APP) (Mf01552291_m1) and peptidylprolyl isomerase B(PPIB) (Mf02802985 ml) Taqman Gene Expression Assays (Thermo FisherScientific).

The relative reduction of APP mRNA was calculated using the comparativecycle threshold (Ct) method. During qPCR, the instrument sets a baselinein the exponential phase of the amplification curve and assigns a Ctvalue based on the intersection point of the baseline with theamplification curve. The APP mRNA reduction was normalized to theexperimental untreated control group as a percentage for each respectivegroup using the Ct values according to the following calculations:ΔCt _(App) =Ct _(App) −CtP _(pib)ΔΔCt _(App) =ΔCt _(App) −ΔCt _(untreated control group mean)Relative mRNA level=2^(−ΔΔCt)

Example 5: Additional RNAi Agent Design, Synthesis, and In VitroScreening in Cos-7, Be(2)-C, and Neuro-2a Cell Lines

This Example describes methods for the design, synthesis, selection, andin vitro screening of additional APP RNAi agents in Cos-7(Dual-Luciferase psiCHECK2 vector), Be(2)-C, and Neuro-2a cells.

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent can be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Cell Culture and Transfections:

Cos-7 cells (ATCC) were transfected by adding 5 μl of 1 ng/μl, dilutedin Opti-MEM, C9orf72 intron 1 psiCHECK2 vector (Blue HeronBiotechnology), 4.9 μl of Opti-MEM plus 0.1 μl of Lipofectamine 2000 perwell (Invitrogen, Carlsbad Calif. cat #11668-019) to 5 μl of siRNAduplexes per well, with 4 replicates of each siRNA duplex, into a384-well plate, and incubated at room temperature for 15 minutes.Thirty-five μl of Dulbecco's Modified Eagle Medium (ThermoFisher)containing ˜5×10³ cells were then added to the siRNA mixture. Cells wereincubated for 48 hours followed by Firefly (transfection control) andRenilla (fused to target sequence) luciferase measurements. Three doseexperiments were performed at 10 nM, 1 nM, and 0.1 nM.

Be(2)-C cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150)to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNAduplex, into a 384-well plate, and incubated at room temperature for 15minutes. Forty μl of 1:1 mixture of Minimum Essential Medium and F12Medium (ThermoFisher) containing ˜5×10³ cells were then added to thesiRNA mixture. Cells were incubated for 48 hours prior to RNApurification. Two dose experiments were performed at 10 nM and 0.1 nM.

Neuro-2a cells (ATCC) were transfected by adding 4.9 μl of Opti-MEM plus0.1 μl of RNAiMAX per well (Invitrogen, Carlsbad Calif. cat #13778-150)to 5 μl of siRNA duplexes per well, with 4 replicates of each siRNAduplex, into a 384-well plate, and incubated at room temperature for 15minutes. Forty μl of Minimum Essential Medium (ThermoFisher) containing˜5×10³ cells were then added to the siRNA mixture. Cells were incubatedfor 48 hours prior to RNA purification. Two dose experiments wereperformed at 10 nM and 0.1 nM.

TOTAL RNA isolation using DYNABEADS mRNA Isolation Kit:

RNA was isolated using an automated protocol on a BioTek-EL406 platformusing DYNABEADs (Invitrogen, cat #61012). Briefly, 70 μl ofLysis/Binding Buffer and 10 μl of lysis buffer containing 3 μl ofmagnetic beads were added to the plate with cells. Plates were incubatedon an electromagnetic shaker for 10 minutes at room temperature and thenmagnetic beads were captured and the supernatant was removed. Bead-boundRNA was then washed 2 times with 150 μl Wash Buffer A and once with WashBuffer B. Beads were then washed with 150 μl Elution Buffer, re-capturedand the supernatant was removed.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

Ten μl of a master mix containing 1 μl 10× Buffer, 0.4 ul 25×dNTPs, 1 μl10× Random primers, 0.5 μl Reverse Transcriptase, 0.5 μl RNase inhibitorand 6.6 μl of H₂O per reaction was added to RNA isolated above. Plateswere sealed, mixed, and incubated on an electromagnetic shaker for 10minutes at room temperature, followed by 2 h 37° C.

Real Time PCR:

Two μl of cDNA and 5 μl Lightcycler 480 probe master mix (Roche Cat#04887301001) were added to either 0.5 μl of Human GAPDH TaqMan Probe(4326317E) and 0.5 μl C9orf72 Human probe (Hs00376619_m1, Thermo) or 0.5μl Mouse GAPDH TaqMan Probe (4352339E) and 0.5 μl C9orf72 Mouse probe(Mm01216837_m1, Thermo) per well in a 384 well plates (Roche cat#04887301001). Real time PCR was done in a LightCycler480 Real Time PCRsystem (Roche). Each duplex was tested at least two times and data werenormalized to cells transfected with a non-targeting control siRNA. Tocalculate relative fold change, real time data were analyzed using theΔΔCt method and normalized to assays performed with cells transfectedwith a non-targeting control siRNA.

Additional APP Oligonucleotide Sequences:

Table 10 through Table16B list additional modified and target APPsequences.

TABLE 10 Additional Human APP Modified Sequences Duplex Sense SEQAntisense SEQ SEQ Name Sequence (5′ to 3′) ID NO Sequence (5′ to 3′)ID NO mRNA Target sequence ID NO AD- asasagagCfaAfAfAfcu 1883asUfscugAfaUfAfguuu 1884 AGAAAGAGCAAAACUAUUCAGAU 1885 506935.2auucagauL96 UfgCfucuuuscsu AD- ususggccAfaCfAfUfga 1886asUfscacUfaAfUfcaug 1887 UCUUGGCCAACAUGAUUAGUGAA 1888 507065.2uuagugauL96 UfuGfgccaasgsa AD- uscsugggUfuGfAfCfaa 1889asUfsugaUfaUfUfuguc 1890 GUUCUGGGUUGACAAAUAUCAAG 1891 507159.2auaucaauL96 AfaCfccagasasc AD- ususuaugAfuUfUfAfcu 1892asGfsauaAfuGfAfguaa 1893 GUUUUAUGAUUUACUCAUUAUCG 1894 507538.2cauuaucuL96 AfuCfauaaasasc AD- asusgccuGfaAfCfUfug 1895asAfsuuaAfuUfCfaagu 1896 AGAUGCCUGAACUUGAAUUAAUC 1897 507624.2aauuaauuL96 UfcAfggcauscsu AD- asgsaugcCfuGfAfAfcu 1898asUfsaauUfcAfAfguuc 1899 GUAGAUGCCUGAACUUGAAUUAA 1900 507724.2ugaauuauL96 AfgGfcaucusasc AD- gscscugaAfcUfUfGfaa 1901asGfsgauUfaAfUfucaa 1902 AUGCCUGAACUUGAAUUAAUCCA 1903 507725.2uuaauccuL96 GfuUfcaggcsasu AD- gsusgguuUfgUfGfAfcc 1904asUfsuaaUfuGfGfguca 1905 UUGUGGUUUGUGACCCAAUUAAG 1906 507789.2caauuaauL96 CfaAfaccacsasa AD- csasgaugCfuUfUfAfga 1907asAfsaauCfuCfUfcuaa 1908 UUCAGAUGCUUUAGAGAGAUUUU 1909 507874.2gagauuuuL96 AfgCfaucugsasa AD- uscsuugcCfuAfAfGfua 1910asAfsaagGfaAfUfacuu 1911 UCUCUUGCCUAAGUAUUCCUUUC 1912 507928.2uuccuuuuL96 AfgGfcaagasgsa AD- ususgcugCfuUTCfUfgc 1913asAfsaauAfuAfGfcaga 1914 GAUUGCUGCUUCUGCUAUAUUUG 1915 507949.2uauauuuuL96 AfgCfagcaasusc Table 10 key: U = uridine-3′-phosphate, u= 2′-O-methyluridine-3′-phosphate, us = 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf = 2′-fluoroguanosine-3′-phosphate, Uf = fluorouridine-3′-phosphate, Cf= 2′-fluorocytidine-3′-phosphate, Af = 2′-fluoroadenosine-3′-phosphate,cs = 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn)= Adenosine-glycol nucleic acid (GNA), gs= 2′-O-methylguanosine-3′-phosphorothioate, (Chd) = 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) =  2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

TABLE 11Additional Human APP Unmodified Sequences; NM_000484.3 and NM_201414.2 TargetingSense SEQ SEQ Duplex Sequence ID Source Name Antisense ID Source NameCross Name (5′ to 3′) NO (Range) Sequence (5′ to 3′) NO (Range) SpeciesAD- AAAGAGCAAAA 1916 NM_000484.3_ AUCUGAAUAGUUUUGCUCU 1917 NM_201414.2_UNK 506935.2 CUAUUCAGAU 1902-1922_s UUCU 1675-1697_as (1902-1922)(1900-1922) AD- UUGGCCAACAU 1918 NM_201414.2_ AUCACUAAUCAUGUUGGCC 1919NM_201414.2_ UNK 507065.2 GAUUAGUGAU 1704-1724_A21U_s AAGA1702-1724_U1A_as (1704-1724) (1702-1724) AD- UCUGGGUUGAC 1920NM_000484.3_ AUUGAUAUUUGUCAACCCA 1921 NM_201414.2_ UNK 507159.2AAAUAUCAAU 2166-2186_G21U_s GAAC 1939-1961_C1A_as (2166-2186)(2164-2186) AD- UUUAUGAUUUA 1922 NM_000484.3_ AGAUAAUGAGUAAAUCAUA 1923NM_201414.2_ UNK 507538.2 CUCAUUAUCU 2613-2633_G21U_s AAAC2386-2408_C1A_as (2613-2633) (2611-2633) AD- AUGCCUGAACU 1924NM_000484.3_ AAUUAAUUCAAGUUCAGGC 1925 NM_201414.2_ UNK 507624.2UGAAUUAAUU 2665-2685_C21U_s AUCU 2438-2460_G1A_as (2665-2685)(2663-2685) AD- AGAUGCCUGAAC 1926 NM_201414.2_ AUAAUUCAAGUUCAGGCAU 1927NM_201414.2_ UNK 507724.2 UUGAAUUAU 2438-2458_A21U_s CUAC2436-2458_U1A_as (2438-2458) (2436-2458) AD- GCCUGAACUUGA 1928NM_201414.2_ AGGAUUAAUUCAAGUUCAG 1929 NM_201414.2_ UNK 507725.2AUUAAUCCU 2442-2462_A21U_s GCAU 2440-2462_U1A_as (2442-2462) (2440-2462)AD- GUGGUUUGUGAC 1930 NM_000484.3_ AUUAAUUGGGUCACAAACC 1931 NM_201414.2_UNK 507789.2 CCAAUUAAU 2853-2873_G21U_s ACAA 2626-2648_C1A_as(2853-2873) (2851-2873) AD- CAGAUGCUUUAG 1932 NM_000484.3_AAAAUCUCUCUAAAGCAUC 1933 NM_201414.2_ UNK 507874.2 AGAGAUUUU 3006-3026_sUGAA 2779-2801_as (3006-3026) (3004-3026) AD- UCUUGCCUAAGU 1934NM_201414.2_ AAAAGGAAUACUUAGGCAA 1935 NM_201414.2_ UNK 507928.2AUUCCUUUU 2718-2738_C21U_s GAGA 2716-2738_G1A_as (2718-2738) (2716-2738)AD- UUGCUGCUUCUG 1936 NM_201414.2_ AAAAUAUAGCAGAAGCAGC 1937 NM_201414.2_UNK 507949.2 CUAUAUUUU 2831-2851_G21U_s AAUC 2829-2851_C1A_as(2831-2851) (2829-2851)

TABLE 12 Additional Human APP Modified Sequences. Duplex Sense SEQAntisense SEQ Name Sequence (5′ to 3′) ID NO Sequence (5′ to 3′) ID NOAD-738012.1 csgscuu(Uhd)CfuAfCfAfcuguauuacaL96 1938VPusGfsuaaUfaCfAfguguAfgAfaagcgsasu 1939 AD-738013.1gscsuuu(Chd)UfaCfAfCfuguauuacaaL96 1940VPusUfsguaAfuAfCfagugUfaGfaaagcsgsa 1941 AD-738014.1ususcua(Chd)AfcUfGfUfauuacauaaaT96 1942VPusUfsuauGfuAfAfuacaGfuGfuagaasasg 1943 AD-738015.1ususucu(Ahd)CfaCfUfGfuauuacauaaL96 1944VPusUfsaugUfaAfUfacagUfgUfagaaasgsc 1945 AD-738016.1asusuua(Ghd)CfuGfUfAfucaaacuagaL96 1946VPusCfsuagUfuUfGfauacAfgCfuaaaususc 1947 AD-738017.1ususccu(Ghd)AfuCfAfCfuaugcauuuaL96 1948VPusAfsaauGfcAfUfagugAfuCfaggaasasg 1949 AD-738018.1gsusgcu(Ghd)UfaAfCfAfcaaguagauaL96 1950VPusAfsucuAfcUfUfguguUfaCfagcacsasg 1951 AD-738019.1ususuag(Chd)UfgUfAfUfcaaacuaguaL96 1952VPusAfscuaGfuUfUfgauaCfaGfcuaaasusu 1953 AD-738020.1ususucc(Uhd)GfaUfCfAfcuaugcauuaT96 1954VPusAfsaugCfaUfAfgugaUfcAfggaaasgsg 1955 AD-738021.1asasugg(Ghd)UfuUfUfGfuguacuguaaL96 1956VPusUfsacaGfuAfCfacaaAfaCfccauusasa 1957 AD-738022.1asusugu(Ahd)CfaGfAfAfucauugcuuaL96 1958VPusAfsagcAfaUfGfauucUfgUfacaauscsa 1959 AD-738023.1ususgua(Chd)AfgAfAfUfcauugcuuaaL96 1960VPusUfsaagCfaAfUfgauuCfuGfuacaasusc 1961 AD-738024.1ususacu(Ghd)UfaCfAfGfauugcugcuaT96 1962VPusAfsgcaGfcAfAfucugUfaCfaguaasasa 1963 AD-738025.1asusaug(Chd)UfgAfAfGfaaguacgucaL96 1964VPusGfsacgUfaCfUfucuuCfaGfcauaususg 1965 AD-738026.1ascscau(Uhd)GfcUfUfCfacuacccauaL96 1966VPusAfsuggGfuAfGfugaaGfcAfauggususu 1967 AD-738027.1csusgug(Chd)UfgUfAfAfcacaaguagaL96 1968VPusCfsuacUfuGfUfguuaCfaGfcacagscsu 1969 AD-738028.1usgscug(Uhd)AfaCfAfCfaaguagaugaL96 1970VPusCfsaucUfaCfUfugugUfuAfcagcascsa 1971 AD-738029.1ascsagc(Uhd)GfuGfCfUfguaacacaaaT96 1972VPusUfsuguGfuUfAfcagcAfcAfgcuguscsa 1973 AD-738030.1gscsugu(Ahd)AfcAfCfAfaguagaugcaL96 1974VPusGfscauCfuAfCfuuguGfuUfacagcsasc 1975 AD-738031.1uscsaaa(Chd)UfaGfUfGfcaugaauagaL96 1976VPusCfsuauUfcAfUfgcacUfaGfuuugasusa 1977 AD-738032.1csasaac(Uhd)AfgUfGfCfaugaauagaaL96 1978VPusUfscuaUfuCfAfugcaCfuAfguuugsasu 1979 AD-738033.1usgscag(Ghd)AfuGfAfUfuguacagaaaL96 1980VPusUfsucuGfuAfCfaaucAfuCfcugcasgsa 1981 AD-738034.1gscsagg(Ahd)UfgAfUfUfguacagaauaT96 1982VPusAfsuucUfgUfAfcaauCfaUfccugcsasg 1983 AD-738035.1csasgga(Uhd)GfaUfUfGfuacagaaucaL96 1984VPusGfsauuCfuGfUfacaaUfcAfuccugscsa 1985 AD-738036.1usasuca(Ahd)AfcUfAfGfugcaugaauaT96 1986VPusAfsuucAfuGfCfacuaGfuUfugauascsa 1987 AD-738037.1ususugu(Ghd)CfcUfGfUfuuuaugugcaL96 1988VPusGfscacAfuAfAfaacaGfgCfacaaasgsa 1989 AD-738038.1ususgug(Chd)CfuGfUfUfuuaugugcaaL96 1990VPusUfsgcaCfaUfAfaaacAfgGfcacaasasg 1991 AD-738039.1csusgca(Ghd)GfaUfGfAfuuguacagaaL96 1992VPusUfscugUfaCfAfaucaUfcCfugcagsasa 1993 AD-738040.1csasggu(Chd)AfuGfAfGfagaaugggaaL96 1994VPusUfscccAfuUfCfucucAfuGfaccugsgsg 1995 AD-738041.1usasugu(Ghd)CfaCfAfCfauuaggcauaL96 1996VPusAfsugcCfuAfAfugugUfgCfacauasasa 1997 AD-738042.1usgsugc(Ahd)CfaCfAfUfuaggcauugaL96 1998VPusCfsaauGfcCfUfaaugUfgUfgcacasusa 1999 AD-738043.1gsgsaug(Ahd)UfuGfUfAfcagaaucauaT96 2000VPusAfsugaUfuCfUfguacAfaUfcauccsusg 2001 AD-738044.1ascscau(Chd)CfaGfAfAfcuggugcaaaL96 2002VPusUfsugcAfcCfAfguucUfgGfaugguscsa 2003 AD-738045.1usasugc(Uhd)GfaAfGfAfaguacguccaL96 2004VPusGfsgacGfuAfCfuucuUfcAfgcauasusu 2005 AD-738046.1asusgcu(Ghd)AfaGfAfAfguacguccgaL96 2006VPusCfsggaCfgUfAfcuucUfuCfagcausasu 2007 AD-738047.1asasacc(Ahd)UfuGfCfUfucacuacccaL96 2008VPusGfsgguAfgUfGfaagcAfaUfgguuususg 2009 AD-738048.1asascca(Uhd)UfgCfUfUfcacuacccaaL96 2010VPusUfsgggUfaGfUfgaagCfaAfugguususu 2011 AD-397217.2csasccg(Ahd)GfaGfAfGfaaugucccaaL96 2012VPusUfsgggAfcAfUfucucUfcUfcggugscsu 2013 AD-738049.1gsusugu(Ahd)UfaUfUfAfuucuuguggaL96 2014VPusCfscacAfaGfAfauaaUfaUfacaacsusg 2015 AD-738050.1ususaug(Uhd)GfcAfCfAfcauuaggcaaL96 2016VPusUfsgccUfaAfUfguguGfcAfcauaasasa 2017 AD-738051.1asusgug(Chd)AfcAfCfAfuuaggcauuaL96 2018VPusAfsaugCfcUfAfauguGfuGfcacausasa 2019 AD-738052.1gsusgca(Chd)AfcAfUfUfaggcauugaaT96 2020VPusUfscaaUfgCfCfuaauGfuGfugcacsasu 2021 AD-738053.1usgsauu(Ghd)UfaCfAfGfaaucauugcaL96 2022VPusGfscaaUfgAfUfucugUfaCfaaucasusc 2023 AD-738054.1gscsuuc(Ahd)CfuAfCfCfcaucgguguaL96 2024VPusAfscacCfgAfUfggguAfgUfgaagcsasa 2025 AD-738055.1ususuua(Uhd)GfuGfCfAfcacauuaggaL96 2026VPusCfscuaAfuGfUfgugcAfcAfuaaaascsa 2027 AD-738056.1csgscuu(Uhd)CfuAfCfAfcuguauuacaL96 2028VPusGfsuaau(Agn)caguguAfgAfaagcgsasu 2029 AD-738057.1gscsuuu(Chd)UfaCfAfCfuguauuacaaL96 2030VPusUfsguaa(Tgn)acagugUfaGfaaagcsgsa 2031 AD-738058.1ususcua(Chd)AfcUfGfUfauuacauaaaT96 2032VPusUfsuaug(Tgn)aauacaGfuGfuagaasasg 2033 AD-738059.1ususucu(Ahd)CfaCfUfGfuauuacauaaL96 2034VPusUfsaugu(Agn)auacagUfgUfagaaasgsc 2035 AD-738060.1asusuua(Ghd)CfuGfUfAfucaaacuagaL96 2036VPusCfsuagu(Tgn)ugauacAfgCfuaaaususc 2037 AD-738061.1ususccu(Ghd)AfuCfAfCfuaugcauuuaL96 2038VPusAfsaaug(Cgn)auagugAfuCfaggaasasg 2039 AD-738062.1gsusgcu(Ghd)UfaAfCfAfcaaguagauaL96 2040VPusAfsucua(Cgn)uuguguUfaCfagcacsasg 2041 AD-738063.1ususuag(Chd)UfgUfAfUfcaaacuaguaL96 2042VPusAfscuag(Tgn)uugauaCfaGfcuaaasusu 2043 AD-738064.1ususucc(Uhd)GfaUfCfAfcuaugcauuaT96 2044VPusAfsaugc(Agn)uagugaUfcAfggaaasgsg 2045 AD-738065.1asasugg(Ghd)UfuUfUfGfuguacuguaaL96 2046VPusUfsacag(Tgn)acacaaAfaCfccauusasa 2047 AD-738066.1ususacu(Ghd)UfaCfAfGfauugcugcuaT96 2048VPusAfsgcag(Cgn)aaucugUfaCfaguaasasa 2049 AD-738067.1asusugu(Ahd)CfaGfAfAfucauugcuuaL96 2050VPusAfsagca(Agn)ugauucUfgUfacaauscsa 2051 AD-738068.1ususgua(Chd)AfgAfAfUfcauugcuuaaL96 2052VPusUfsaagc(Agn)augauuCfuGfuacaasusc 2053 AD-738069.1asusaug(Chd)UfgAfAfGfaaguacgucaL96 2054VPusGfsacgu(Agn)cuucuuCfaGfcauaususg 2055 AD-738070.1ascscau(Uhd)GfcUfUfCfacuacccauaL96 2056VPusAfsuggg(Tgn)agugaaGfcAfauggususu 2057 AD-738071.1csusgug(Chd)UfgUfAfAfcacaaguagaL96 2058VPusCfsuacu(Tgn)guguuaCfaGfcacagscsu 2059 AD-738072.1usgscug(Uhd)AfaCfAfCfaaguagaugaL96 2060VPusCfsaucu(Agn)cuugugUfuAfcagcascsa 2061 AD-738073.1ascsagc(Uhd)GfuGfCfUfguaacacaaaT96 2062VPusUfsugug(Tgn)uacagcAfcAfgcuguscsa 2063 AD-738074.1gscsugu(Ahd)AfcAfCfAfaguagaugcaL96 2064VPusGfscauc(Tgn)acuuguGfuUfacagcsasc 2065 AD-738075.1uscsaaa(Chd)UfaGfUfGfcaugaauagaL96 2066VPusCfsuauu(Cgn)augcacUfaGfuuugasusa 2067 AD-738076.1csasaac(Uhd)AfgUfGfCfaugaauagaaL96 2068VPusUfscuau(Tgn)caugcaCfuAfguuugsasu 2069 AD-738077.1usgscag(Ghd)AfuGfAfUfuguacagaaaL96 2070VPusUfsucug(Tgn)acaaucAfuCfcugcasgsa 2071 AD-738078.1gscsagg(Ahd)UfgAfUfUfguacagaauaT96 2072VPusAfsuucu(Ggn)uacaauCfaUfccugcsasg 2073 AD-738079.1csasgga(Uhd)GfaUfUfGfuacagaaucaL96 2074VPusGfsauuc(Tgn)guacaaUfcAfuccugscsa 2075 AD-738080.1usasuca(Ahd)AfcUfAfGfugcaugaauaT96 2076VPusAfsuuca(Tgn)gcacuaGfuUfugauascsa 2077 AD-738081.1ususugu(Ghd)CfcUfGfUfuuuaugugcaL96 2078VPusGfscaca(Tgn)aaaacaGfgCfacaaasgsa 2079 AD-738082.1ususgug(Chd)CfuGfUfUfuuaugugcaaL96 2080VPusUfsgcac(Agn)uaaaacAfgGfcacaasasg 2081 AD-738083.1csusgca(Ghd)GfaUfGfAfuuguacagaaL96 2082VPusUfscugu(Agn)caaucaUfcCfugcagsasa 2083 AD-738084.1csasggu(Chd)AfuGfAfGfagaaugggaaL96 2084VPusUfsccca(Tgn)ucucucAfuGfaccugsgsg 2085 AD-738085.1usasugc(Uhd)GfaAfGfAfaguacguccaL96 2086VPusGfsgacg(Tgn)acuucuUfcAfgcauasusu 2087 AD-738086.1asusgcu(Ghd)AfaGfAfAfguacguccgaL96 2088VPusCfsggac(Ggn)uacuucUfuCfagcausasu 2089 AD-738087.1asasacc(Ahd)UfuGfCfUfucacuacccaL96 2090VPusGfsggua(Ggn)ugaagcAfaUfgguuususg 2091 AD-738088.1asascca(Uhd)UfgCfUfUfcacuacccaaL96 2092VPusUfsgggu(Agn)gugaagCfaAfugguususu 2093 AD-738089.1usasugu(Ghd)CfaCfAfCfauuaggcauaL96 2094VPusAfsugcc(Tgn)aaugugUfgCfacauasasa 2095 AD-738090.1usgsugc(Ahd)CfaCfAfUfuaggcauugaL96 2096VPusCfsaaug(Cgn)cuaaugUfgUfgcacasusa 2097 AD-738091.1gsgsaug(Ahd)UfuGfUfAfcagaaucauaT96 2098VPusAfsugau(Tgn)cuguacAfaUfcauccsusg 2099 AD-738092.1ascscau(Chd)CfaGfAfAfcuggugcaaaL96 2100VPusUfsugca(Cgn)caguucUfgGfaugguscsa 2101 AD-738093.1csasccg(Ahd)GfaGfAfGfaaugucccaaL96 2102VPusUfsggga(Cgn)auucucUfcUfcggugscsu 2103 AD-738094.1gsusugu(Ahd)UfaUfUfAfuucuuguggaL96 2104VPusCfscaca(Agn)gaauaaUfaUfacaacsusg 2105 AD-738095.1ususaug(Uhd)GfcAfCfAfcauuaggcaaL96 2106VPusUfsgccu(Agn)auguguGfcAfcauaasasa 2107 AD-738096.1asusgug(Chd)AfcAfCfAfuuaggcauuaL96 2108VPusAfsaugc(Cgn)uaauguGfuGfcacausasa 2109 AD-738097.1gsusgca(Chd)AfcAfUfUfaggcauugaaT96 2110VPusUfscaau(Ggn)ccuaauGfuGfugcacsasu 2111 AD-738098.1usgsauu(Ghd)UfaCfAfGfaaucauugcaL96 2112VPusGfscaau(Ggn)auucugUfaCfaaucasusc 2113 AD-738099.1gscsuuc(Ahd)CfuAfCfCfcaucgguguaL96 2114VPusAfscacc(Ggn)auggguAfgUfgaagcsasa 2115 AD-738100.1ususuua(Uhd)GfuGfCfAfcacauuaggaL96 2116VPusCfscuaa(Tgn)gugugcAfcAfuaaaascsa 2117 Table 12 key: U= uridine-3′-phosphate, u = 2′-O-methyluridine-3′-phosphate, us = 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf = 2′-fluoroguanosine-3′-phosphate, Uf = fluorouridine-3′-phosphate, Cf= 2′-fluorocytidine-3′-phosphate, Af = 2′-fluoroadenosine-3′-phosphate,cs = 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn)= Adenosine-glycol nucleic acid (GNA), gs= 2′-O-methylguanosine-3′-phosphorothioate, (Chd) = 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) =  2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

TABLE 13 Additional Human APP Unmodified Sequences;XM_005548887.2 and NM_001198823.1 Targeting. Duplex Sense SEQ AntisenseSEQ Name Sequence (5′ to 3′) ID NO Sequence (5′ to 3′) ID NO Source NameAD- CGCUUUCUACACUGUAUUACA 2118 UGUAAUACAGUGUAGAAAGCGAU 2119XM_005548887.2_ 738012.1 3401-3423_as AD- GCUUUCUACACUGUAUUACAA 2120UUGUAAUACAGUGUAGAAAGCGA 2121 XM_005548887.2_ 738013.1 3402-3424_as AD-UUCUACACUGUAUUACAUAAA 2122 UUUAUGUAAUACAGUGUAGAAAG 2123 NM_001198823.1_738014.1 3306-3328_as AD- UUUCUACACUGUAUUACAUAA 2124UUAUGUAAUACAGUGUAGAAAGC 2125 NM_001198823.1_ 738015.1 3305-3327_as AD-AUUUAGCUGUAUCAAACUAGA 2126 UCUAGUUUGAUACAGCUAAAUUC 2127 XM_005548887.2_738016.1 2837-2859_as AD- UUCCUGAUCACUAUGCAUUUA 2128UAAAUGCAUAGUGAUCAGGAAAG 2129 XM_005548887.2_ 738017.1 3030-3052_as AD-GUGCUGUAACACAAGUAGAUA 2130 UAUCUACUUGUGUUACAGCACAG 2131 NM_001198823.1_738018.1 2602-2624_C1A_as AD- UUUAGCUGUAUCAAACUAGUA 2132UACUAGUUUGAUACAGCUAAAUU 2133 XM_005548887.2_ 738019.1 2838-2860_as AD-UUUCCUGAUCACUAUGCAUUA 2134 UAAUGCAUAGUGAUCAGGAAAGG 2135 XM_005548887.2_738020.1 3029-3051_as AD- AAUGGGUUUUGUGUACUGUAA 2136UUACAGUACACAAAACCCAUUAA 2137 XM_005548887.2_ 738021.1 2813-2835_as AD-AUUGUACAGAAUCAUUGCUUA 2138 UAAGCAAUGAUUCUGUACAAUCA 2139 NM_001198823.1_738022.1 3272-3294_as AD- UUGUACAGAAUCAUUGCUUAA 2140UUAAGCAAUGAUUCUGUACAAUC 2141 NM_001198823.1_ 738023.1 3273-3295_as AD-UUACUGUACAGAUUGCUGCUA 2142 UAGCAGCAAUCUGUACAGUAAAA 2143 XM_005548887.2_738024.1 3113-3135_as AD- AUAUGCUGAAGAAGUACGUCA 2144UGACGUACUUCUUCAGCAUAUUG 2145 XM_005548887.2_ 738025.1 1740-1762_as AD-ACCAUUGCUUCACUACCCAUA 2146 UAUGGGUAGUGAAGCAAUGGUUU 2147 NM_001198823.1_738026.1 2506-2528_G1A_as AD- CUGUGCUGUAACACAAGUAGA 2148UCUACUUGUGUUACAGCACAGCU 2149 NM_001198823.1_ 738027.1 2600-2622_as AD-UGCUGUAACACAAGUAGAUGA 2150 UCAUCUACUUGUGUUACAGCACA 2151 NM_001198823.1_738028.1 2603-2625_G1A_as AD- ACAGCUGUGCUGUAACACAAA 2152UUUGUGUUACAGCACAGCUGUCA 2153 NM_001198823.1_ 738029.1 2596-2618_C1A_asAD- GCUGUAACACAAGUAGAUGCA 2154 UGCAUCUACUUGUGUUACAGCAC 2155NM_001198823.1_ 738030.1 2604-2626_G1A_as AD- UCAAACUAGUGCAUGAAUAGA 2156UCUAUUCAUGCACUAGUUUGAUA 2157 NM_001198823.1_ 738031.1 2742-2764_as AD-CAAACUAGUGCAUGAAUAGAA 2158 UUCUAUUCAUGCACUAGUUUGAU 2159 NM_001198823.1_738032.1 2743-2765_as AD- UGCAGGAUGAUUGUACAGAAA 2160UUUCUGUACAAUCAUCCUGCAGA 2161 NM_001198823.1_ 738033.1 3263-3285_as AD-GCAGGAUGAUUGUACAGAAUA 2162 UAUUCUGUACAAUCAUCCUGCAG 2163 NM_001198823.1_738034.1 3264-3286_G1A_as AD- CAGGAUGAUUGUACAGAAUCA 2164UGAUUCUGUACAAUCAUCCUGCA 2165 NM_001198823.1_ 738035.1 3265-3287_as AD-UAUCAAACUAGUGCAUGAAUA 2166 UAUUCAUGCACUAGUUUGAUACA 2167 NM_001198823.1_738036.1 2740-2762_as AD- UUUGUGCCUGUUUUAUGUGCA 2168UGCACAUAAAACAGGCACAAAGA 2169 NM_001198823.1_ 738037.1 3070-3092_as AD-UUGUGCCUGUUUUAUGUGCAA 2170 UUGCACAUAAAACAGGCACAAAG 2171 NM_001198823.1_738038.1 3071-3093_G1A_as AD- CUGCAGGAUGAUUGUACAGAA 2172UUCUGUACAAUCAUCCUGCAGAA 2173 NM_001198823.1_ 738039.1 3262-3284_as AD-CAGGUCAUGAGAGAAUGGGAA 2174 UUCCCAUUCUCUCAUGACCUGGG 2175 NM_001198823.1_738040.1 1369-1391_as AD- UAUGUGCACACAUUAGGCAUA 2176UAUGCCUAAUGUGUGCACAUAAA 2177 NM_001198823.1_ 738041.1 3083-3105_as AD-UGUGCACACAUUAGGCAUUGA 2178 UCAAUGCCUAAUGUGUGCACAUA 2179 NM_001198823.1_738042.1 3085-3107_as AD- GGAUGAUUGUACAGAAUCAUA 2180UAUGAUUCUGUACAAUCAUCCUG 2181 NM_001198823.1_ 738043.1 3267-3289_as AD-ACCAUCCAGAACUGGUGCAAA 2182 UUUGCACCAGUUCUGGAUGGUCA 2183 NM_001198823.1_738044.1 424-446_C1A_as AD- UAUGCUGAAGAAGUACGUCCA 2184UGGACGUACUUCUUCAGCAUAUU 2185 XM_005548887.2_ 738045.1 1741-1763_as AD-AUGCUGAAGAAGUACGUCCGA 2186 UCGGACGUACUUCUUCAGCAUAU 2187 XM_005548887.2_738046.1 1742-1764_as AD- AAACCAUUGCUUCACUACCCA 2188UGGGUAGUGAAGCAAUGGUUUUG 2189 XM_005548887.2_ 738047.1 2614-2636_as AD-AACCAUUGCUUCACUACCCAA 2190 UUGGGUAGUGAAGCAAUGGUUUU 2191 XM_005548887.2_738048.1 2615-2637_as AD- CACCGAGAGAGAAUGUCCCAA 2192UUGGGACAUUCUCUCUCGGUGCU 2193 NM_001198823.1_ 397217.2 1351-1373_C1A_asAD- GUUGUAUAUUAUUCUUGUGGA 2194 UCCACAAGAAUAAUAUACAACUG 2195XM_005548887.2_ 738049.1 2906-2928_as AD- UUAUGUGCACACAUUAGGCAA 2196UUGCCUAAUGUGUGCACAUAAAA 2197 NM_001198823.1_ 738050.1 3082-3104_as AD-AUGUGCACACAUUAGGCAUUA 2198 UAAUGCCUAAUGUGUGCACAUAA 2199 NM_001198823.1_738051.1 3084-3106_C1A_as AD- GUGCACACAUUAGGCAUUGAA 2200UUCAAUGCCUAAUGUGUGCACAU 2201 NM_001198823.1_ 738052.1 3086-3108_C1A_asAD- UGAUUGUACAGAAUCAUUGCA 2202 UGCAAUGAUUCUGUACAAUCAUC 2203NM_001198823.1_ 738053.1 3270-3292_as AD- GCUUCACUACCCAUCGGUGUA 2204UACACCGAUGGGUAGUGAAGCAA 2205 NM_001198823.1_ 738054.1 2512-2534_as AD-UUUUAUGUGCACACAUUAGGA 2206 UCCUAAUGUGUGCACAUAAAACA 2207 NM_001198823.1_738055.1 3080-3102_G1A_as AD- CGCUUUCUACACUGUAUUACA 2208UGUAAUACAGUGUAGAAAGCGAU 2209 XM_005548887.2_ 738056.1 3401-3423_as AD-GCUUUCUACACUGUAUUACAA 2210 UUGUAATACAGUGUAGAAAGCGA 2211 XM_005548887.2_738057.1 3402-3424_as AD- UUCUACACUGUAUUACAUAAA 2212UUUAUGTAAUACAGUGUAGAAAG 2213 XM_005548887.2_ 738058.1 3405-3427_as AD-UUUCUACACUGUAUUACAUAA 2214 UUAUGUAAUACAGUGUAGAAAGC 2215 XM_005548887.2_738059.1 3404-3426_as AD- AUUUAGCUGUAUCAAACUAGA 2216UCUAGUTUGAUACAGCUAAAUUC 2217 XM_005548887.2_ 738060.1 2837-2859_as AD-UUCCUGAUCACUAUGCAUUUA 2218 UAAAUGCAUAGUGAUCAGGAAAG 2219 XM_005548887.2_738061.1 3030-3052_as AD- GUGCUGUAACACAAGUAGAUA 2220UAUCUACUUGUGUUACAGCACAG 2221 XM_005548887.2_ 738062.1 2716-2738_as AD-UUUAGCUGUAUCAAACUAGUA 2222 UACUAGTUUGAUACAGCUAAAUU 2223 XM_005548887.2_738063.1 2838-2860_as AD- UUUCCUGAUCACUAUGCAUUA 2224UAAUGCAUAGUGAUCAGGAAAGG 2225 XM_005548887.2_ 738064.1 3029-3051_as AD-AAUGGGUUUUGUGUACUGUAA 2226 UUACAGTACACAAAACCCAUUAA 2227 XM_005548887.2_738065.1 2813-2835_as AD- UUACUGUACAGAUUGCUGCUA 2228UAGCAGCAAUCUGUACAGUAAAA 2229 XM_005548887.2_ 738066.1 3113-3135_as AD-AUUGUACAGAAUCAUUGCUUA 2230 UAAGCAAUGAUUCUGUACAAUCA 2231 XM_005548887.2_738067.1 3371-3393_as AD- UUGUACAGAAUCAUUGCUUAA 2232UUAAGCAAUGAUUCUGUACAAUC 2233 XM_005548887.2_ 738068.1 3372-3394_as AD-AUAUGCUGAAGAAGUACGUCA 2234 UGACGUACUUCUUCAGCAUAUUG 2235 XM_005548887.2_738069.1 1740-1762_as AD- ACCAUUGCUUCACUACCCAUA 2236UAUGGGTAGUGAAGCAAUGGUUU 2237 XM_005548887.2_ 738070.1 2616-2638_as AD-CUGUGCUGUAACACAAGUAGA 2238 UCUACUTGUGUUACAGCACAGCU 2239 XM_005548887.2_738071.1 2714-2736_as AD- UGCUGUAACACAAGUAGAUGA 2240UCAUCUACUUGUGUUACAGCACA 2241 XM_005548887.2_ 738072.1 2717-2739_as AD-ACAGCUGUGCUGUAACACAAA 2242 UUUGUGTUACAGCACAGCUGUCA 2243 XM_005548887.2_738073.1 2710-2732_as AD- GCUGUAACACAAGUAGAUGCA 2244UGCAUCTACUUGUGUUACAGCAC 2245 XM_005548887.2_ 738074.1 2718-2740_as AD-UCAAACUAGUGCAUGAAUAGA 2246 UCUAUUCAUGCACUAGUUUGAUA 2247 XM_005548887.2_738075.1 2848-2870_as AD- CAAACUAGUGCAUGAAUAGAA 2248UUCUAUTCAUGCACUAGUUUGAU 2249 XM_005548887.2_ 738076.1 2849-2871_as AD-UGCAGGAUGAUUGUACAGAAA 2250 UUUCUGTACAAUCAUCCUGCAGA 2251 XM_005548887.2_738077.1 3362-3384_as AD- GCAGGAUGAUUGUACAGAAUA 2252UAUUCUGUACAAUCAUCCUGCAG 2253 XM_005548887.2_ 738078.1 3363-3385_as AD-CAGGAUGAUUGUACAGAAUCA 2254 UGAUUCTGUACAAUCAUCCUGCA 2255 XM_005548887.2_738079.1 3364-3386_as AD- UAUCAAACUAGUGCAUGAAUA 2256UAUUCATGCACUAGUUUGAUACA 2257 XM_005548887.2_ 738080.1 2846-2868_as AD-UUUGUGCCUGUUUUAUGUGCA 2258 UGCACATAAAACAGGCACAAAGA 2259 XM_005548887.2_738081.1 3180-3202_as AD- UUGUGCCUGUUUUAUGUGCAA 2260UUGCACAUAAAACAGGCACAAAG 2261 XM_005548887.2_ 738082.1 3181-3203_as AD-CUGCAGGAUGAUUGUACAGAA 2262 UUCUGUACAAUCAUCCUGCAGAA 2263 XM_005548887.2_738083.1 3361-3383_as AD- CAGGUCAUGAGAGAAUGGGAA 2264UUCCCATUCUCUCAUGACCUGGG 2265 XM_005548887.2_ 738084.1 1487-1509_as AD-UAUGCUGAAGAAGUACGUCCA 2266 UGGACGTACUUCUUCAGCAUAUU 2267 XM_005548887.2_738085.1 1741-1763_as AD- AUGCUGAAGAAGUACGUCCGA 2268UCGGACGUACUUCUUCAGCAUAU 2269 XM_005548887.2_ 738086.1 1742-1764_as AD-AAACCAUUGCUUCACUACCCA 2270 UGGGUAGUGAAGCAAUGGUUUUG 2271 XM_005548887.2_738087.1 2614-2636_as AD- AACCAUUGCUUCACUACCCAA 2272UUGGGUAGUGAAGCAAUGGUUUU 2273 XM_005548887.2_ 738088.1 2615-2637_as AD-UAUGUGCACACAUUAGGCAUA 2274 UAUGCCTAAUGUGUGCACAUAAA 2275 XM_005548887.2_738089.1 3193-3215_as AD- UGUGCACACAUUAGGCAUUGA 2276UCAAUGCCUAAUGUGUGCACAUA 2277 XM_005548887.2_ 738090.1 3195-3217_as AD-GGAUGAUUGUACAGAAUCAUA 2278 UAUGAUTCUGUACAAUCAUCCUG 2279 XM_005548887.2_738091.1 3366-3388_as AD- ACCAUCCAGAACUGGUGCAAA 2280UUUGCACCAGUUCUGGAUGGUCA 2281 XM_005548887.2_ 738092.1 767-789_as AD-CACCGAGAGAGAAUGUCCCAA 2282 UUGGGACAUUCUCUCUCGGUGCU 2283 XM_005548887.2_738093.1 1469-1491_as AD- GUUGUAUAUUAUUCUUGUGGA 2284UCCACAAGAAUAAUAUACAACUG 2285 XM_005548887.2_ 738094.1 2906-2928_as AD-UUAUGUGCACACAUUAGGCAA 2286 UUGCCUAAUGUGUGCACAUAAAA 2287 XM_005548887.2_738095.1 3192-3214_as AD- AUGUGCACACAUUAGGCAUUA 2288UAAUGCCUAAUGUGUGCACAUAA 2289 XM_005548887.2_ 738096.1 3194-3216_as AD-GUGCACACAUUAGGCAUUGAA 2290 UUCAAUGCCUAAUGUGUGCACAU 2291 XM_005548887.2_738097.1 3196-3218_as AD- UGAUUGUACAGAAUCAUUGCA 2292UGCAAUGAUUCUGUACAAUCAUC 2293 XM_005548887.2_ 738098.1 3369-3391_as AD-GCUUCACUACCCAUCGGUGUA 2294 UACACCGAUGGGUAGUGAAGCAA 2295 XM_005548887.2_738099.1 2622-2644_as AD- UUUUAUGUGCACACAUUAGGA 2296UCCUAATGUGUGCACAUAAAACA 2297 XM_005548887.2_ 738100.1 3190-3212_as

TABLE 14 Additional Human APP Modified Sequences. SEQ SEQ Tar- Duplex IDID get Name Sense Sequence (5′ to 3′) NO Antisense Sequence (5′ to 3′)NO APP AD- usasgug(Chd)AfuGfAfAfuagauucucaL96 2298VPusGfsagaa(Tgn)cuauucAfuGfcacuasgsu 2299 886823.1 APP AD-usasgug(Chd)AfugAfAfuagauucucaL96 2300VPusGfsagaa(Tgn)cuauucAfuGfcacuasgsu 2301 886824.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2302VPusGfsagaa(Tgn)cuauucAfuGfcacuasgsu 2303 886825.1 APP AD-usasgug(Chd)AfudGadAuagauucucaL96 2304VPusGfsagaa(Tgn)cuaulfcAfuGfcacuasgsu 2305 886826.1 APP AD-usasgug(Chd)AfuGfAfAfuagauucucaL96 2306VPusdGsagaa(Tgn)cuauucAfuGfcacuasgsu 2307 886827.1 APP AD-usasgug(Chd)AfuGfAfAfuagauucucaL96 2308VPusGfsagaa(Tgn)cuauucAfugcacuasgsu 2309 886828.1 APP AD-usasgug(Chd)AfuGfAfAfuagauucucaL96 2310VPusGfsagaa(Tgn)cuauucAfudGcacuasgsu 2311 886829.1 APP AD-usasgug(Chd)AfuGfaAfuagauucucaL96 2312VPusGfsagaa(Tgn)cuaudTcAfudGcacuasgsu 2313 886830.1 APP AD-usasgug(Chd)AfuGfaAfuagauucucaL96 2314VPusGfsagaa(Tgn)cuaudTcAfugcacuasgsu 2315 886831.1 APP AD-usasgug(Chd)AfuGfAfAfuagauucucaL96 2316VPusdGsagaa(Tgn)cuauucAfudGcacuasgsu 2317 886832.1 APP AD-usasgug(Chd)AfuGfaAfuagauucucaL96 2318VPusdGsagaa(Tgn)cuaudTcAfudGcacuasgsu 2319 886833.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2320VPusdGsagaa(Tgn)cuauucAfuGfcacuasgsu 2321 886834.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2322VPusGfsagaa(Tgn)cuauucAfudGcacuasgsu 2323 886836.1 APP AD-usasgug(Chd)AfudGaAfuagauucucaL96 2324VPusGfsagaa(Tgn)cuaudTcAfudGcacuasgsu 2325 886837.1 APP AD-usasgug(Chd)AfudGaAfuagauucucaL96 2326VPusGfsagaa(Tgn)cuaudTcAfugcacuasgsu 2327 886838.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2328VPusdGsagaa(Tgn)cuauucAfudGcacuasgsu 2329 886839.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2330VPusdGsagaa(Tgn)cuauucAfudGcacuasgsu 2331 886839.2 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2332VPusdGsagaa(Tgn)cuaudTcAfudGcacuasgsu 2333 886840.1 APP AD-usasgug(Chd)AfudGaAfuagauucucaL96 2334VPusdGsagaa(Tgn)cuaudTcAfudGcacuasgsu 2335 886841.1 APP AD-usasgug(Chd)AfudGadAuagauucucaL96 2336VPusdGsagaa(Tgn)cuaudTcAfudGcacuasgsu 2337 886842.1 APP AD-usasgug(Chd)audGadAuagauucucaL96 2338VPusdGsagaa(Tgn)cuaudTcAfudGcacuasgsu 2339 886843.1 APP AD-usasgug(Chd)audGadAuagauucucaL96 2340VPudGagaa(Tgn)cuaudTcAfudGcacuasgsu 2341 886844.1 APP AD-usasgug(Chd)audGadAuagauucucaL96 2342VPudGagaa(Tgn)cuauUfcAfudGcacuasgsu 2343 886845.1 APP AD-usasgug(Chd)audGadAuagauucucaL96 2344 VPudGadGadAucuauUfcAfudGcacuasgsu2345 886846.1 APP AD- usasgug(Chd)AfudGAfAfuagauucucaL96 2346VPudGagaa(Tgn)cuauucAfudGcacuasgsu 2347 886847.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2348VPusdGsagadA(Tgn)cuauucAfudGcacuasgsu 2349 886848.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2350VPusdGsagdAa(Tgn)cuauucAfudGcacuasgsu 2351 886849.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2352VPusdGsagadA(Tgn)cuaudTcAfudGcacuasgsu 2353 886850.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2354VPusdGsagdAa(Tgn)cuaudTcAfudGcacuasgsu 2355 886851.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2356VPusdGsagadA(Tgn)cuaudTcAfugcacuasgsu 2357 886852.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2358VPusdGsagdAa(Tgn)cuaudTcAfugcacuasgsu 2359 886853.1 APP AD-usasgug(Chd)AfudGadAuagauucucaL96 2360VPusdGsagdAa(Tgn)cuaudTcAfugcacuasgsu 2361 886854.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2362VPudGagadA(Tgn)cuauucAfudGcacuasgsu 2363 886855.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2364VPudGagdAa(Tgn)cuauucAfudGcacuasgsu 2365 886856.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2366VPudGagadA(Tgn)cuaudTcAfudGcacuasgsu 2367 886857.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2368VPudGagdAa(Tgn)cuaudTcAfudGcacuasgsu 2369 886858.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2370VPudGagadA(Tgn)cuaudTcAfugcacuasgsu 2371 886859.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2372VPudGagdAa(Tgn)cuaudTcAfugcacuasgsu 2373 886860.1 APP AD-usasgug(Chd)AfuGfAfAfuagauucucaL96 2374VPusGfsagaa(Tgn)cuauucAfuGfcacuasusg 2375 886861.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2376VPusdGsagadA(Tgn)cuaudTcAfudGcacuasusg 2377 886862.1 APP AD-usasgug(Chd)AfudGAfAfuagauucucaL96 2378VPusdGsagdAa(Tgn)cuaudTcAfudGcacuasusg 2379 886863.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2380VPusUfsaggu(Tgn)ggauuuUfcGfuagccsgsu 2381 886864.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2382VPusUfsaggu(Tgn)ggauuuUfcGfuagccsgsu 2383 886865.1 APP AD-gsgscua(Chd)dGaAfAfAfuccaaccuaaL96 2384VPusUfsaggu(Tgn)ggauuuUfcGfuagccsgsu 2385 886866.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2386VPuUfaggu(Tgn)ggauuuUfcGfuagccsgsu 2387 886867.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2388VPusUfsaggu(Tgn)ggauuuUfcguagccsgsu 2389 886868.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2390VPusUfsaggu(Tgn)ggauuutlfcdGuagccsgsu 2391 886869.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2392VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu 2393 886870.1 APP AD-gsgscua(Chd)gaAfaAfuccaaccuaaL96 2394VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu 2395 886871.1 APP AD-gsgscua(Chd)gaAfaAfuccaaccuaaL96 2396VPusUfsaggu(Tgn)ggauUfuUfcdGuagccsgsu 2397 886872.1 APP AD-gsgscua(Chd)gadAadAuccaaccuaaL96 2398VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu 2399 886873.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2400VPusUfsaggu(Tgn)ggaudTuUfcguagccsgsu 2401 886874.1 APP AD-gsgscua(Chd)gaAfaAfuccaaccuaaL96 2402VPusUfsaggu(Tgn)ggaudTuUfcguagccsgsu 2403 886875.1 APP AD-gsgscua(Chd)gaAfaAfuccaaccuaaL96 2404VPusUfsaggu(Tgn)ggauUfuUfcguagccsgsu 2405 886876.1 APP AD-gsgscua(Chd)gadAadAuccaaccuaaL96 2406VPusUfsaggu(Tgn)ggaudTuUfcguagccsgsu 2407 886877.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2408VPusUfsaggu(Tgn)ggauuuUfcguagccsgsu 2409 886878.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2410VPusUfsaggu(Tgn)ggauuutlfcdGuagccsgsu 2411 886879.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2412VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu 2413 886880.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2414VPusUfsaggu(Tgn)ggaudTuUfcguagccsgsu 2415 886881.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2416VPuUfaggdT(Tgn)ggauuuUfcguagccsgsu 2417 886882.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2418VPuUfaggdT(Tgn)ggauuuUTcdGuagccsgsu 2419 886883.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2420VPuUfaggdT(Tgn)ggaudTuUfcdGuagccsgsu 2421 886884.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2422VPuUfaggdT(Tgn)ggaudTuUfcguagccsgsu 2423 886885.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2424VPuUfagdGu(Tgn)ggauuufacguagccsgsu 2425 886886.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2426VPuUfagdGu(Tgn)ggauuufacdGuagccsgsu 2427 886887.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2428VPuUfagdGu(Tgn)ggaudTuUfcdGuagccsgsu 2429 886888.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2430VPuUfagdGu(Tgn)ggaudTuUfcguagccsgsu 2431 886889.1 APP AD-gsgscua(Chd)GfaAfAfAfuccaaccuaaT96 2432VPusUfsaggu(Tgn)ggauuufacGfuagccsusg 2433 886890.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2434VPusUfsaggu(Tgn)ggauuufacdGuagccsusg 2435 886891.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2436VPuUfaggdT(Tgn)ggaudTuUfcdGuagccsusg 2437 886892.1 APP AD-gsgscua(Chd)gaAfAfAfuccaaccuaaL96 2438VPuUfagdGu(Tgn)ggaudTuUfcdGuagccsusg 2439 886893.1 APP AD-asasaga(Ghd)CfaAfAfAfcuauucagaaL96 2440VPusUfscugAfaUfAfguuuUfgCfucuuuscsu 2441 886894.1 APP AD-asasag(Ahd)gCfaAfAfAfcuauucagaaT96 2442VPusUfscugAfaUfAfguuuUfgCfucuuuscsu 2443 886895.1 APP AD-asasagag(Chd)aAfAfAfcuauucagaaL96 2444VPusUfscugAfaUfAfguuuUfgCfucuuuscsu 2445 886896.1 APP AD-asasagagCfaAfAfAfcua(Uhd)ucagaaL96 2446VPusUfscugAfaUfAfguuuUfgCfucuuuscsu 2447 886897.1 APP AD-asasagagCfaAfAfAfcuau(Uhd)cagaaT96 2448VPusUfscugAfaUfAfguuuUfgCfucuuuscsu 2449 886898.1 APP AD-asasagagCfaAfAfAfcuauu(Chd)agaaL96 2450VPusUfscugAfaUfAfguuuUfgCfucuuuscsu 2451 886899.1 APP AD-asasaga(Ghd)CfaAfAfAfcuauucagaaL96 2452VPusUfscugAfauaguuuUfgCfucuuuscsu 2453 886900.1 APP AD-asasaga(Ghd)CfaAfAfAfcuauucagaaL96 2454VPuUfcugAfaUfAfguuuUfgCfucuuuscsu 2455 886901.1 APP AD-asasaga(Ghd)CfaAfAfAfcuauucagaaL96 2456 VPuUfcugAfauaguuuUfgCfucuuuscsu2457 886902.1 APP AD- asasag(Ahd)gCfaAfAfAfcuauucagaaT96 2458VPuUfcugAfaUfAfguuuUfgCfucuuuscsu 2459 886903.1 APP AD-asasag(Ahd)gCfaAfAfAfcuauucagaaT96 2460 VPuUfcugAfauaguuuUfgCfucuuuscsu2461 886904.1 APP AD- asasagag(Chd)aAfAfAfcuauucagaaL96 2462VPuUfcugAfaUfAfguuuUfgCfucuuuscsu 2463 886905.1 APP AD-asasagag(Chd)aAfAfAfcuauucagaaL96 2464 VPuUfcugAfauaguuuUfgCfucuuuscsu2465 886906.1 APP AD- asasagag(Chd)aAfaAfcuauucagaaL96 2466VPuUfcugAfauagudTuUfgCfucuuuscsu 2467 886907.1 APP AD-asasagag(Chd)adAadAcuauucagaaL96 2468 VPuUfcugAfauagudTuUfgCfucuuuscsu2469 886908.1 APP AD- asasagag(Chd)adAadAcuauucagaaL96 2470VPuUfcugdAauagudTuUfgdCucuuuscsu 2471 886909.1 APP AD-asasaga(Ghd)CfaAfAfAfcuauucagaaL96 2472VPusUfscugAfauaguuuUfgCfucuuususg 2473 886910.1 APP AD-asasagagCfaAfAfAfcua(Uhd)ucagaaL96 2474 VPuUfcugAfauaguuuUfgCfucuuususg2475 886911.1 APP AD- asasagag(Chd)aAfAfAfcuauucagaaL96 2476VPuUfcugAfauaguuuUfgCfucuuususg 2477 886912.1 APP AD-ususuau(Ghd)AfuUfUfAfcucauuaucaL96 2478VPusGfsauaAfuGfAfguaaAfuCfauaaasasc 2479 886913.1 APP AD-ususua(Uhd)gAfuUfUfAfcucauuaucaL96 2480VPusGfsauaAfuGfAfguaaAfuCfauaaasasc 2481 886914.1 APP AD-ususuaug(Ahd)uUfUfAfcucauuaucaL96 2482VPusGfsauaAfuGfAfguaaAfuCfauaaasasc 2483 886915.1 APP AD-ususuaugAfuUTUfAfcuc(Ahd)uuaucaL96 2484VPusGfsauaAfuGfAfguaaAfuCfauaaasasc 2485 886916.1 APP AD-ususuaugAfuUfUfAfcuca(Uhd)uaucaL96 2486VPusGfsauaAfuGfAfguaaAfuCfauaaasasc 2487 886917.1 APP AD-ususuaugAfuUfUfAfcucau(Uhd)aucaL96 2488VPusGfsauaAfuGfAfguaaAfuCfauaaasasc 2489 886918.1 APP AD-ususuau(Ghd)AfuUfUfAfcucauuaucaL96 2490VPusGfsauaAfugaguaaAfuCfauaaasasc 2491 886919.1 APP AD-ususuau(Ghd)AfuUfUfAfcucauuaucaL96 2492VPusdGsauaAfugaguaaAfuCfauaaasasc 2493 886920.1 APP AD-ususuau(Ghd)AfuUfUfAfcucauuaucaL96 2494 VPudGauaAfugaguaaAfuCfauaaasasc2495 886921.1 APP AD- ususua(Uhd)gAfuUfUfAfcucauuaucaL96 2496VPusdGsauaAfugaguaaAfuCfauaaasasc 2497 886922.1 APP AD-ususua(Uhd)gAfuUfUfAfcucauuaucaL96 2498 VPudGauaAfugaguaaAfuCfauaaasasc2499 886923.1 APP AD- ususuaug(Ahd)uUfUfAfcucauuaucaL96 2500VPusdGsauaAfugaguaaAfuCfauaaasasc 2501 886924.1 APP AD-ususuaug(Ahd)uUfUfAfcucauuaucaL96 2502 VPudGauaAfugaguaaAfuCfauaaasasc2503 886925.1 APP AD- ususuaug(Ahd)uUfuAfcucauuaucaL96 2504VPudGauadAugagudAaAfuCfauaaasasc 2505 886926.1 APP AD-ususuaug(Ahd)uUfudAcucauuaucaL96 2506 VPudGauadAugagudAaAfuCfauaaasasc2507 886927.1 APP AD- ususuaug(Ahd)uUfudAcucauuaucaL96 2508VPudGauadAugagudAaAfudCauaaasasc 2509 886928.1 APP AD-ususuau(Ghd)AfuUfUfAfcucauuaucaL96 2510VPusGfsauaAfugaguaaAfuCfauaaasusg 2511 886929.1 APP AD-ususuaugAfuUTUfAfcuc(Ahd)uuaucaL96 2512VPusdGsauaAfugaguaaAfuCfauaaasusg 2513 886930.1 APP AD-ususuaug(Ahd)uUfUfAfcucauuaucaL96 2514 VPusdGsauaAfugaguaaAfuCfauaaasusg2515 886931.1 Table 14 key: U = uridine-3′-phosphate, u= 2′-O-methyluridine-3′-phosphate, us = 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf = 2′-fluoroguanosine-3′-phosphate, Uf = fluorouridine-3′-phosphate, Cf= 2′-fluorocytidine-3′-phosphate, Af = 2′-fluoroadenosine-3′-phosphate,cs = 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn)= Adenosine-glycol nucleic acid (GNA), gs= 2′-O-methylguanosine-3′-phosphorothioate, (Chd) = 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) =  2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

TABLE 15  Additional APP Unmodified Sequences. Duplex Sense Sequence SEQ ID Antisense Sequence  SEQ Name (5′ to 3′) NO (5′ to 3′) ID NO AD-UAGUGCAUGAAUAGAUUCUCA 2516 UGAGAATCUAUUCAUGCACUAGU 2517 886823.1 AD-UAGUGCAUGAAUAGAUUCUCA 2518 UGAGAATCUAUUCAUGCACUAGU 2519 886824.1 AD-UAGUGCAUGAAUAGAUUCUCA 2520 UGAGAATCUAUUCAUGCACUAGU 2521 886825.1 AD-UAGUGCAUGAAUAGAUUCUCA 2522 UGAGAATCUAUUCAUGCACUAGU 2523 886826.1 AD-UAGUGCAUGAAUAGAUUCUCA 2524 UGAGAATCUAUUCAUGCACUAGU 2525 886827.1 AD-UAGUGCAUGAAUAGAUUCUCA 2526 UGAGAATCUAUUCAUGCACUAGU 2527 886828.1 AD-UAGUGCAUGAAUAGAUUCUCA 2528 UGAGAATCUAUUCAUGCACUAGU 2529 886829.1 AD-UAGUGCAUGAAUAGAUUCUCA 2530 UGAGAATCUAUTCAUGCACUAGU 2531 886830.1 AD-UAGUGCAUGAAUAGAUUCUCA 2532 UGAGAATCUAUTCAUGCACUAGU 2533 886831.1 AD-UAGUGCAUGAAUAGAUUCUCA 2534 UGAGAATCUAUUCAUGCACUAGU 2535 886832.1 AD-UAGUGCAUGAAUAGAUUCUCA 2536 UGAGAATCUAUTCAUGCACUAGU 2537 886833.1 AD-UAGUGCAUGAAUAGAUUCUCA 2538 UGAGAATCUAUUCAUGCACUAGU 2539 886834.1 AD-UAGUGCAUGAAUAGAUUCUCA 2540 UGAGAATCUAUUCAUGCACUAGU 2541 886836.1 AD-UAGUGCAUGAAUAGAUUCUCA 2542 UGAGAATCUAUTCAUGCACUAGU 2543 886837.1 AD-UAGUGCAUGAAUAGAUUCUCA 2544 UGAGAATCUAUTCAUGCACUAGU 2545 886838.1 AD-UAGUGCAUGAAUAGAUUCUCA 2546 UGAGAATCUAUUCAUGCACUAGU 2547 886839.1 AD-UAGUGCAUGAAUAGAUUCUCA 2548 UGAGAATCUAUUCAUGCACUAGU 2549 886839.2 AD-UAGUGCAUGAAUAGAUUCUCA 2550 UGAGAATCUAUTCAUGCACUAGU 2551 886840.1 AD-UAGUGCAUGAAUAGAUUCUCA 2552 UGAGAATCUAUTCAUGCACUAGU 2553 886841.1 AD-UAGUGCAUGAAUAGAUUCUCA 2554 UGAGAATCUAUTCAUGCACUAGU 2555 886842.1 AD-UAGUGCAUGAAUAGAUUCUCA 2556 UGAGAATCUAUTCAUGCACUAGU 2557 886843.1 AD-UAGUGCAUGAAUAGAUUCUCA 2558 UGAGAATCUAUTCAUGCACUAGU 2559 886844.1 AD-UAGUGCAUGAAUAGAUUCUCA 2560 UGAGAATCUAUUCAUGCACUAGU 2561 886845.1 AD-UAGUGCAUGAAUAGAUUCUCA 2562 UGAGAAUCUAUUCAUGCACUAGU 2563 886846.1 AD-UAGUGCAUGAAUAGAUUCUCA 2564 UGAGAATCUAUUCAUGCACUAGU 2565 886847.1 AD-UAGUGCAUGAAUAGAUUCUCA 2566 UGAGAATCUAUUCAUGCACUAGU 2567 886848.1 AD-UAGUGCAUGAAUAGAUUCUCA 2568 UGAGAATCUAUUCAUGCACUAGU 2569 886849.1 AD-UAGUGCAUGAAUAGAUUCUCA 2570 UGAGAATCUAUTCAUGCACUAGU 2571 886850.1 AD-UAGUGCAUGAAUAGAUUCUCA 2572 UGAGAATCUAUTCAUGCACUAGU 2573 886851.1 AD-UAGUGCAUGAAUAGAUUCUCA 2574 UGAGAATCUAUTCAUGCACUAGU 2575 886852.1 AD-UAGUGCAUGAAUAGAUUCUCA 2576 UGAGAATCUAUTCAUGCACUAGU 2577 886853.1 AD-UAGUGCAUGAAUAGAUUCUCA 2578 UGAGAATCUAUTCAUGCACUAGU 2579 886854.1 AD-UAGUGCAUGAAUAGAUUCUCA 2580 UGAGAATCUAUUCAUGCACUAGU 2581 886855.1 AD-UAGUGCAUGAAUAGAUUCUCA 2582 UGAGAATCUAUUCAUGCACUAGU 2583 886856.1 AD-UAGUGCAUGAAUAGAUUCUCA 2584 UGAGAATCUAUTCAUGCACUAGU 2585 886857.1 AD-UAGUGCAUGAAUAGAUUCUCA 2586 UGAGAATCUAUTCAUGCACUAGU 2587 886858.1 AD-UAGUGCAUGAAUAGAUUCUCA 2588 UGAGAATCUAUTCAUGCACUAGU 2589 886859.1 AD-UAGUGCAUGAAUAGAUUCUCA 2590 UGAGAATCUAUTCAUGCACUAGU 2591 886860.1 AD-UAGUGCAUGAAUAGAUUCUCA 2592 UGAGAATCUAUUCAUGCACUAUG 2593 886861.1 AD-UAGUGCAUGAAUAGAUUCUCA 2594 UGAGAATCUAUTCAUGCACUAUG 2595 886862.1 AD-UAGUGCAUGAAUAGAUUCUCA 2596 UGAGAATCUAUTCAUGCACUAUG 2597 886863.1 AD-GGCUACGAAAAUCCAACCUAA 2598 UUAGGUTGGAUUUUCGUAGCCGU 2599 886864.1 AD-GGCUACGAAAAUCCAACCUAA 2600 UUAGGUTGGAUUUUCGUAGCCGU 2601 886865.1 AD-GGCUACGAAAAUCCAACCUAA 2602 UUAGGUTGGAUUUUCGUAGCCGU 2603 886866.1 AD-GGCUACGAAAAUCCAACCUAA 2604 UUAGGUTGGAUUUUCGUAGCCGU 2605 886867.1 AD-GGCUACGAAAAUCCAACCUAA 2606 UUAGGUTGGAUUUUCGUAGCCGU 2607 886868.1 AD-GGCUACGAAAAUCCAACCUAA 2608 UUAGGUTGGAUUUUCGUAGCCGU 2609 886869.1 AD-GGCUACGAAAAUCCAACCUAA 2610 UUAGGUTGGAUTUUCGUAGCCGU 2611 886870.1 AD-GGCUACGAAAAUCCAACCUAA 2612 UUAGGUTGGAUTUUCGUAGCCGU 2613 886871.1 AD-GGCUACGAAAAUCCAACCUAA 2614 UUAGGUTGGAUUUUCGUAGCCGU 2615 886872.1 AD-GGCUACGAAAAUCCAACCUAA 2616 UUAGGUTGGAUTUUCGUAGCCGU 2617 886873.1 AD-GGCUACGAAAAUCCAACCUAA 2618 UUAGGUTGGAUTUUCGUAGCCGU 2619 886874.1 AD-GGCUACGAAAAUCCAACCUAA 2620 UUAGGUTGGAUTUUCGUAGCCGU 2621 886875.1 AD-GGCUACGAAAAUCCAACCUAA 2622 UUAGGUTGGAUUUUCGUAGCCGU 2623 886876.1 AD-GGCUACGAAAAUCCAACCUAA 2624 UUAGGUTGGAUTUUCGUAGCCGU 2625 886877.1 AD-GGCUACGAAAAUCCAACCUAA 2626 UUAGGUTGGAUUUUCGUAGCCGU 2627 886878.1 AD-GGCUACGAAAAUCCAACCUAA 2628 UUAGGUTGGAUUUUCGUAGCCGU 2629 886879.1 AD-GGCUACGAAAAUCCAACCUAA 2630 UUAGGUTGGAUTUUCGUAGCCGU 2631 886880.1 AD-GGCUACGAAAAUCCAACCUAA 2632 UUAGGUTGGAUTUUCGUAGCCGU 2633 886881.1 AD-GGCUACGAAAAUCCAACCUAA 2634 UUAGGTTGGAUUUUCGUAGCCGU 2635 886882.1 AD-GGCUACGAAAAUCCAACCUAA 2636 UUAGGTTGGAUUUUCGUAGCCGU 2637 886883.1 AD-GGCUACGAAAAUCCAACCUAA 2638 UUAGGTTGGAUTUUCGUAGCCGU 2639 886884.1 AD-GGCUACGAAAAUCCAACCUAA 2640 UUAGGTTGGAUTUUCGUAGCCGU 2641 886885.1 AD-GGCUACGAAAAUCCAACCUAA 2642 UUAGGUTGGAUUUUCGUAGCCGU 2643 886886.1 AD-GGCUACGAAAAUCCAACCUAA 2644 UUAGGUTGGAUUUUCGUAGCCGU 2645 886887.1 AD-GGCUACGAAAAUCCAACCUAA 2646 UUAGGUTGGAUTUUCGUAGCCGU 2647 886888.1 AD-GGCUACGAAAAUCCAACCUAA 2648 UUAGGUTGGAUTUUCGUAGCCGU 2649 886889.1 AD-GGCUACGAAAAUCCAACCUAA 2650 UUAGGUTGGAUUUUCGUAGCCUG 2651 886890.1 AD-GGCUACGAAAAUCCAACCUAA 2652 UUAGGUTGGAUUUUCGUAGCCUG 2653 886891.1 AD-GGCUACGAAAAUCCAACCUAA 2654 UUAGGTTGGAUTUUCGUAGCCUG 2655 886892.1 AD-GGCUACGAAAAUCCAACCUAA 2656 UUAGGUTGGAUTUUCGUAGCCUG 2657 886893.1 AD-AAAGAGCAAAACUAUUCAGAA 2658 UUCUGAAUAGUUUUGCUCUUUCU 2659 886894.1 AD-AAAGAGCAAAACUAUUCAGAA 2660 UUCUGAAUAGUUUUGCUCUUUCU 2661 886895.1 AD-AAAGAGCAAAACUAUUCAGAA 2662 UUCUGAAUAGUUUUGCUCUUUCU 2663 886896.1 AD-AAAGAGCAAAACUAUUCAGAA 2664 UUCUGAAUAGUUUUGCUCUUUCU 2665 886897.1 AD-AAAGAGCAAAACUAUUCAGAA 2666 UUCUGAAUAGUUUUGCUCUUUCU 2667 886898.1 AD-AAAGAGCAAAACUAUUCAGAA 2668 UUCUGAAUAGUUUUGCUCUUUCU 2669 886899.1 AD-AAAGAGCAAAACUAUUCAGAA 2670 UUCUGAAUAGUUUUGCUCUUUCU 2671 886900.1 AD-AAAGAGCAAAACUAUUCAGAA 2672 UUCUGAAUAGUUUUGCUCUUUCU 2673 886901.1 AD-AAAGAGCAAAACUAUUCAGAA 2674 UUCUGAAUAGUUUUGCUCUUUCU 2675 886902.1 AD-AAAGAGCAAAACUAUUCAGAA 2676 UUCUGAAUAGUUUUGCUCUUUCU 2677 886903.1 AD-AAAGAGCAAAACUAUUCAGAA 2678 UUCUGAAUAGUUUUGCUCUUUCU 2679 886904.1 AD-AAAGAGCAAAACUAUUCAGAA 2680 UUCUGAAUAGUUUUGCUCUUUCU 2681 886905.1 AD-AAAGAGCAAAACUAUUCAGAA 2682 UUCUGAAUAGUUUUGCUCUUUCU 2683 886906.1 AD-AAAGAGCAAAACUAUUCAGAA 2684 UUCUGAAUAGUTUUGCUCUUUCU 2685 886907.1 AD-AAAGAGCAAAACUAUUCAGAA 2686 UUCUGAAUAGUTUUGCUCUUUCU 2687 886908.1 AD-AAAGAGCAAAACUAUUCAGAA 2688 UUCUGAAUAGUTUUGCUCUUUCU 2689 886909.1 AD-AAAGAGCAAAACUAUUCAGAA 2690 UUCUGAAUAGUUUUGCUCUUUUG 2691 886910.1 AD-AAAGAGCAAAACUAUUCAGAA 2692 UUCUGAAUAGUUUUGCUCUUUUG 2693 886911.1 AD-AAAGAGCAAAACUAUUCAGAA 2694 UUCUGAAUAGUUUUGCUCUUUUG 2695 886912.1 AD-UUUAUGAUUUACUCAUUAUCA 2696 UGAUAAUGAGUAAAUCAUAAAAC 2697 886913.1 AD-UUUAUGAUUUACUCAUUAUCA 2698 UGAUAAUGAGUAAAUCAUAAAAC 2699 886914.1 AD-UUUAUGAUUUACUCAUUAUCA 2700 UGAUAAUGAGUAAAUCAUAAAAC 2701 886915.1 AD-UUUAUGAUUUACUCAUUAUCA 2702 UGAUAAUGAGUAAAUCAUAAAAC 2703 886916.1 AD-UUUAUGAUUUACUCAUUAUCA 2704 UGAUAAUGAGUAAAUCAUAAAAC 2705 886917.1 AD-UUUAUGAUUUACUCAUUAUCA 2706 UGAUAAUGAGUAAAUCAUAAAAC 2707 886918.1 AD-UUUAUGAUUUACUCAUUAUCA 2708 UGAUAAUGAGUAAAUCAUAAAAC 2709 886919.1 AD-UUUAUGAUUUACUCAUUAUCA 2710 UGAUAAUGAGUAAAUCAUAAAAC 2711 886920.1 AD-UUUAUGAUUUACUCAUUAUCA 2712 UGAUAAUGAGUAAAUCAUAAAAC 2713 886921.1 AD-UUUAUGAUUUACUCAUUAUCA 2714 UGAUAAUGAGUAAAUCAUAAAAC 2715 886922.1 AD-UUUAUGAUUUACUCAUUAUCA 2716 UGAUAAUGAGUAAAUCAUAAAAC 2717 886923.1 AD-UUUAUGAUUUACUCAUUAUCA 2718 UGAUAAUGAGUAAAUCAUAAAAC 2719 886924.1 AD-UUUAUGAUUUACUCAUUAUCA 2720 UGAUAAUGAGUAAAUCAUAAAAC 2721 886925.1 AD-UUUAUGAUUUACUCAUUAUCA 2722 UGAUAAUGAGUAAAUCAUAAAAC 2723 886926.1 AD-UUUAUGAUUUACUCAUUAUCA 2724 UGAUAAUGAGUAAAUCAUAAAAC 2725 886927.1 AD-UUUAUGAUUUACUCAUUAUCA 2726 UGAUAAUGAGUAAAUCAUAAAAC 2727 886928.1 AD-UUUAUGAUUUACUCAUUAUCA 2728 UGAUAAUGAGUAAAUCAUAAAUG 2729 886929.1 AD-UUUAUGAUUUACUCAUUAUCA 2730 UGAUAAUGAGUAAAUCAUAAAUG 2731 886930.1 AD-UUUAUGAUUUACUCAUUAUCA 2732 UGAUAAUGAGUAAAUCAUAAAUG 2733 886931.1

TABLE 16A  Additional Human APP Modified and Unmodified Sense Sequences.Modified Unmodified Duplex Sense Sequence  SEQ ID Sense Sequence  SEQ IDName target (5′ to 3′) NO (5′ to 3′) NO AD-961583 APP gsgscua(Chd)gadA2734 GGCUACGAAAA 2735 adAuccaaccusasa UCCAACCUAA AD-961584 APPasasagag(Chd)aAf 2736 AAAGAGCAAAA 2737 aAfcuauucagsasa CUAUUCAGAAAD-961585 APP asasagag(Chd)adA 2738 AAAGAGCAAAA 2739 adAcuauucagsasaCUAUUCAGAA AD-961586 APP ususuau(Ghd)AfuUa 2740 UUUAUGAUUUA 2741fUfAfcucuuauscsa CUCAUUAUCA Table 16A key: U = uridine-3′-phosphate, u= 2′-O-methyluridine-3′-phosphate, us= 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf= 2′-fluoroguanosine-3′-phosphate, Uf = 2′-fluorouridine-3′-phosphate,Cf = 2′-fluorocytidine-3′-phosphate, Af= 2′-fluoroadenosine-3′-phosphate, cs = 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn) = Adenosine-glycol nucleic acid (GNA),gs = 2′-O-methylguanosine-3′-phosphorothioate, (Chd)= 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) = 2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

TABLE 16B  Additional Human APP Modified and Unmodified Antisense Sequences Duplex Modified Antisense  SEQ ID Unmodified Antisense  SEQ ID Name target Sequence (5′ to 3′) NOSequence (5′ to 3′) NO AD-961583 APP VPusUfsaggu(Tgn) 2742 UUAGGUTGGAUT2743 ggaudTuUfcdGuagc UUCGUAGCCGU csgsu AD-961584 APP VPuUfcugAfauagud2744 UUCUGAAUAGUT 2745 TuUfgCfucuuuscsu UUGCUCUUUCU AD-961585 APPVPuUfcugdAauagud 2746 UUCUGAAUAGUT 2747 TuUfgdCucuuuscsu UUGCUCUUUCUAD-961586 APP VPusGfsauaAfugagu 2748 UGAUAAUGAGUA 2749 aaAfuCfauaaasusgAAUCAUAAAUG Table 16B key: U = uridine-3′-phosphate, u= 2′-O-methyluridine-3′-phosphate, us= 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf= 2′-fluoroguanosine-3′-phosphate, Uf = 2′-fluorouridine-3′-phosphate,Cf = 2′-fluorocytidine-3′-phosphate, Af= 2′-fluoroadenosine-3′-phosphate, cs= 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn)= Adenosine-glycol nucleic acid (GNA), gs= 2′-O-methylguanosine-3′-phosphorothioate, (Chd)= 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) = 2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

Table 17 summarizes results from a multi-dose APP screen in Be(2) cellsconducted at either 10 nM, 1 nM or 0.1 nM. Data are expressed as percentmessage remaining relative to AD-1955 non-targeting control.

TABLE 17 APP Dose Screen Study in Be(2)C Cell Lines at 10 nM, 1 nM, and0.1 nM Average message Standard Duplex remaining (%) Deviation Dose DoseUnit AD-738012.1 12.47 3.92 10 nM AD-738013.1 8.78 1.74 10 nMAD-738014.1 10.27 3.95 10 nM AD-738015.1 9.84 3.00 10 nM AD-738016.111.79 4.10 10 nM AD-738017.1 12.85 2.41 10 nM AD-738018.1 13.22 2.40 10nM AD-738019.1 14.57 2.64 10 nM AD-738020.1 9.06 2.84 10 nM AD-738021.112.95 6.42 10 nM AD-738022.1 10.55 1.29 10 nM AD-738023.1 8.22 1.41 10nM AD-738024.1 13.51 4.75 10 nM AD-738025.1 48.96 7.46 10 nM AD-738026.111.78 2.88 10 nM AD-738027.1 10.71 2.22 10 nM AD-738028.1 18.52 2.12 10nM AD-738029.1 17.74 4.49 10 nM AD-738030.1 25.60 5.77 10 nM AD-738031.128.70 6.14 10 nM AD-738032.1 13.38 9.34 10 nM AD-738033.1 10.13 1.96 10nM AD-738034.1 15.22 6.91 10 nM AD-738035.1 14.59 5.75 10 nM AD-738036.119.64 12.56 10 nM AD-738037.1 21.74 10.22 10 nM AD-738038.1 27.23 3.7310 nM AD-738039.1 28.08 5.99 10 nM AD-738040.1 60.35 0.96 10 nMAD-738041.1 38.29 15.92 10 nM AD-738042.1 25.54 7.15 10 nM AD-738043.112.59 4.84 10 nM AD-738044.1 44.57 13.69 10 nM AD-738045.1 218.56 104.8310 nM AD-738046.1 263.77 29.64 10 nM AD-738047.1 35.84 3.46 10 nMAD-738048.1 34.43 4.01 10 nM AD-397217.2 70.05 6.00 10 nM AD-738049.113.20 6.16 10 nM AD-738050.1 11.02 0.82 10 nM AD-738051.1 40.85 6.01 10nM AD-738052.1 37.45 14.43 10 nM AD-738053.1 30.69 7.50 10 nMAD-738054.1 62.81 13.33 10 nM AD-738055.1 28.18 9.27 10 nM AD-738056.128.91 4.29 10 nM AD-738057.1 24.47 7.91 10 nM AD-738058.1 49.05 8.41 10nM AD-738059.1 35.32 9.27 10 nM AD-738060.1 25.40 3.87 10 nM AD-738061.153.19 2.95 10 nM AD-738062.1 17.28 7.65 10 nM AD-738063.1 33.40 9.94 10nM AD-738064.1 30.75 4.43 10 nM AD-738065.1 28.34 14.64 10 nMAD-738066.1 92.51 16.17 10 nM AD-738067.1 30.74 7.71 10 nM AD-738068.125.12 2.84 10 nM AD-738069.1 59.72 9.34 10 nM AD-738070.1 35.03 9.43 10nM AD-738071.1 15.79 2.79 10 nM AD-738072.1 63.54 33.06 10 nMAD-738073.1 28.05 3.62 10 nM AD-738074.1 31.74 5.88 10 nM AD-738075.1174.04 56.95 10 nM AD-738076.1 29.35 8.89 10 nM AD-738077.1 14.69 5.0010 nM AD-738078.1 15.15 2.61 10 nM AD-738079.1 11.40 3.42 10 nMAD-738080.1 10.80 0.91 10 nM AD-738081.1 36.37 8.31 10 nM AD-738082.128.65 4.80 10 nM AD-738083.1 9.98 0.75 10 nM AD-738084.1 31.76 4.26 10nM AD-738085.1 48.74 6.11 10 nM AD-738086.1 60.41 10.30 10 nMAD-738087.1 12.21 2.15 10 nM AD-738088.1 44.49 10.16 10 nM AD-738089.131.43 4.82 10 nM AD-738090.1 23.34 5.54 10 nM AD-738091.1 35.28 12.92 10nM AD-738092.1 89.59 18.72 10 nM AD-738093.1 71.33 16.07 10 nMAD-738094.1 18.69 3.23 10 nM AD-738095.1 30.93 6.90 10 nM AD-738096.126.70 5.20 10 nM AD-738097.1 65.74 9.99 10 nM AD-738098.1 16.18 4.17 10nM AD-738099.1 48.95 9.69 10 nM AD-738100.1 67.26 11.31 10 nMAD-738012.1 17.40 2.53 1 nM AD-738013.1 15.51 2.70 1 nM AD-738014.123.54 9.95 1 nM AD-738015.1 21.35 2.38 1 nM AD-738016.1 20.20 1.90 1 nMAD-738017.1 15.67 2.60 1 nM AD-738018.1 17.00 0.80 1 nM AD-738019.117.58 7.97 1 nM AD-738020.1 15.47 3.64 1 nM AD-738021.1 14.81 4.24 1 nMAD-738022.1 13.71 2.86 1 nM AD-738023.1 17.33 4.91 1 nM AD-738024.120.64 7.04 1 nM AD-738025.1 95.81 28.98 1 nM AD-738026.1 28.29 10.28 1nM AD-738027.1 15.94 3.44 1 nM AD-738028.1 25.76 10.62 1 nM AD-738029.118.83 6.50 1 nM AD-738030.1 30.24 7.29 1 nM AD-738031.1 30.77 6.54 1 nMAD-738032.1 25.98 6.57 1 nM AD-738033.1 31.28 8.14 1 nM AD-738034.125.06 6.27 1 nM AD-738035.1 21.67 1.11 1 nM AD-738036.1 32.29 11.81 1 nMAD-738037.1 30.77 5.48 1 nM AD-738038.1 19.03 1.00 1 nM AD-738039.120.25 5.55 1 nM AD-738040.1 51.87 7.09 1 nM AD-738041.1 35.67 8.23 1 nMAD-738042.1 33.70 9.34 1 nM AD-738043.1 19.76 3.35 1 nM AD-738044.143.40 9.46 1 nM AD-738045.1 97.99 13.43 1 nM AD-738046.1 112.65 25.09 1nM AD-738047.1 37.50 4.18 1 nM AD-738048.1 23.67 0.94 1 nM AD-397217.260.11 7.67 1 nM AD-738049.1 20.00 1.41 1 nM AD-738050.1 36.49 7.06 1 nMAD-738051.1 27.03 6.08 1 nM AD-738052.1 31.82 7.17 1 nM AD-738053.114.96 2.91 1 nM AD-738054.1 32.00 5.62 1 nM AD-738055.1 27.57 7.73 1 nMAD-738056.1 15.16 0.70 1 nM AD-738057.1 14.83 3.32 1 nM AD-738058.133.09 9.91 1 nM AD-738059.1 26.76 5.77 1 nM AD-738060.1 11.79 2.64 1 nMAD-738061.1 28.49 1.35 1 nM AD-738062.1 15.89 6.49 1 nM AD-738063.125.01 8.31 1 nM AD-738064.1 16.91 2.56 1 nM AD-738065.1 15.45 2.85 1 nMAD-738066.1 51.85 8.48 1 nM AD-738067.1 20.90 4.96 1 nM AD-738068.115.82 2.70 1 nM AD-738069.1 81.26 2.84 1 nM AD-738070.1 59.48 11.42 1 nMAD-738071.1 15.12 3.89 1 nM AD-738072.1 40.16 7.78 1 nM AD-738073.118.46 5.20 1 nM AD-738074.1 27.74 1.97 1 nM AD-738075.1 83.53 9.94 1 nMAD-738076.1 50.62 3.51 1 nM AD-738077.1 21.52 4.49 1 nM AD-738078.124.49 10.05 1 nM AD-738079.1 8.66 2.69 1 nM AD-738080.1 28.88 1.12 1 nMAD-738081.1 77.35 10.22 1 nM AD-738082.1 48.10 10.63 1 nM AD-738083.123.74 4.60 1 nM AD-738084.1 100.84 2.83 1 nM AD-738085.1 101.30 4.73 1nM AD-738086.1 60.29 24.33 1 nM AD-738087.1 9.71 3.71 1 nM AD-738088.179.16 7.79 1 nM AD-738089.1 35.37 8.78 1 nM AD-738090.1 37.16 13.37 1 nMAD-738091.1 49.56 10.83 1 nM AD-738092.1 79.50 10.15 1 nM AD-738093.196.42 16.26 1 nM AD-738094.1 41.63 5.90 1 nM AD-738095.1 45.03 8.10 1 nMAD-738096.1 44.52 11.55 1 nM AD-738097.1 78.88 13.42 1 nM AD-738098.128.84 8.43 1 nM AD-738099.1 68.10 16.73 1 nM AD-738100.1 84.53 5.73 1 nMAD-738012.1 35.64 12.05 0.1 nM AD-738013.1 29.76 5.05 0.1 nM AD-738014.147.17 13.55 0.1 nM AD-738015.1 35.51 13.38 0.1 nM AD-738016.1 38.17 9.760.1 nM AD-738017.1 30.03 7.04 0.1 nM AD-738018.1 20.38 4.76 0.1 nMAD-738019.1 30.10 4.89 0.1 nM AD-738020.1 44.67 8.48 0.1 nM AD-738021.130.05 5.88 0.1 nM AD-738022.1 30.24 5.96 0.1 nM AD-738023.1 25.74 7.750.1 nM AD-738024.1 31.43 10.51 0.1 nM AD-738025.1 112.57 14.24 0.1 nMAD-738026.1 54.28 6.70 0.1 nM AD-738027.1 26.02 4.95 0.1 nM AD-738028.135.82 10.41 0.1 nM AD-738029.1 40.29 3.76 0.1 nM AD-738030.1 51.38 24.040.1 nM AD-738031.1 40.78 11.79 0.1 nM AD-738032.1 47.97 6.74 0.1 nMAD-738033.1 38.57 7.04 0.1 nM AD-738034.1 46.53 13.21 0.1 nM AD-738035.143.04 12.39 0.1 nM AD-738036.1 43.08 3.41 0.1 nM AD-738037.1 87.09 39.320.1 nM AD-738038.1 64.97 3.06 0.1 nM AD-738039.1 74.15 30.96 0.1 nMAD-738040.1 159.41 39.34 0.1 nM AD-738041.1 108.29 36.98 0.1 nMAD-738042.1 69.15 28.46 0.1 nM AD-738043.1 45.00 17.66 0.1 nMAD-738044.1 88.04 17.84 0.1 nM AD-738045.1 238.11 15.24 0.1 nMAD-738046.1 259.68 3.44 0.1 nM AD-738047.1 136.91 44.65 0.1 nMAD-738048.1 131.72 13.39 0.1 nM AD-397217.2 222.75 51.71 0.1 nMAD-738049.1 65.58 6.12 0.1 nM AD-738050.1 63.97 11.64 0.1 nM AD-738051.189.72 27.54 0.1 nM AD-738052.1 140.07 36.18 0.1 nM AD-738053.1 77.0914.75 0.1 nM AD-738054.1 205.91 46.37 0.1 nM AD-738055.1 197.02 44.700.1 nM AD-738056.1 85.09 14.19 0.1 nM AD-738057.1 87.72 18.23 0.1 nMAD-738058.1 164.40 24.71 0.1 nM AD-738059.1 129.01 9.61 0.1 nMAD-738060.1 63.48 35.21 0.1 nM AD-738061.1 191.48 13.85 0.1 nMAD-738062.1 108.14 8.70 0.1 nM AD-738063.1 100.27 16.53 0.1 nMAD-738064.1 46.78 12.88 0.1 nM AD-738065.1 84.72 11.97 0.1 nMAD-738066.1 218.00 48.39 0.1 nM AD-738067.1 123.65 34.39 0.1 nMAD-738068.1 90.93 17.12 0.1 nM AD-738069.1 300.08 12.73 0.1 nMAD-738070.1 238.24 7.61 0.1 nM AD-738071.1 46.50 1.25 0.1 nM AD-738072.158.01 21.95 0.1 nM AD-738073.1 68.05 19.98 0.1 nM AD-738074.1 134.7730.73 0.1 nM AD-738075.1 328.84 50.48 0.1 nM AD-738076.1 237.89 30.070.1 nM AD-738077.1 108.45 14.70 0.1 nM AD-738078.1 127.49 44.03 0.1 nMAD-738079.1 46.06 9.44 0.1 nM AD-738080.1 57.45 19.09 0.1 nM AD-738081.1147.89 27.56 0.1 nM AD-738082.1 169.52 28.01 0.1 nM AD-738083.1 106.746.93 0.1 nM AD-738084.1 242.62 60.78 0.1 nM AD-738085.1 295.62 32.59 0.1nM AD-738086.1 221.56 21.04 0.1 nM AD-738087.1 82.58 14.78 0.1 nMAD-738088.1 88.52 10.41 0.1 nM AD-738089.1 84.36 20.12 0.1 nMAD-738090.1 120.67 19.87 0.1 nM AD-738091.1 180.61 14.25 0.1 nMAD-738092.1 240.22 16.63 0.1 nM AD-738093.1 303.63 8.82 0.1 nMAD-738094.1 146.42 25.16 0.1 nM AD-738095.1 124.16 57.91 0.1 nMAD-738096.1 56.53 8.58 0.1 nM AD-738097.1 116.46 38.97 0.1 nMAD-738098.1 59.28 19.71 0.1 nM AD-738099.1 149.49 42.85 0.1 nMAD-738100.1 89.06 17.49 0.1 nM

Table 18 summarizes results from a multi-dose APP screen in Neuro2Acells conducted at either 10 nM, 1 nM or 0.1 nM. Data are expressed aspercent message remaining relative to AD-1955 non-targeting control

TABLE 18 APP Dose Screen Study in Neuro2A Cell Lines at 10 nM, 1 nM, and0.1 nM. Standard Duplex Average Deviation Dose Dose Unit AD-738012.10.11 0.07 10 nM AD-738013.1 0.20 0.06 10 nM AD-738014.1 1.12 0.42 10 nMAD-738015.1 1.72 1.20 10 nM AD-738016.1 0.98 0.31 10 nM AD-738017.1 0.320.24 10 nM AD-738018.1 0.14 0.07 10 nM AD-738019.1 0.63 0.25 10 nMAD-738020.1 0.11 0.08 10 nM AD-738021.1 1.20 0.52 10 nM AD-738022.1 1.860.95 10 nM AD-738023.1 1.18 0.53 10 nM AD-738024.1 3.13 1.81 10 nMAD-738025.1 11.77 3.21 10 nM AD-738026.1 0.81 0.44 10 nM AD-738027.10.23 0.10 10 nM AD-738028.1 0.15 0.15 10 nM AD-738029.1 1.48 0.93 10 nMAD-738030.1 1.45 0.99 10 nM AD-738031.1 2.72 0.68 10 nM AD-738032.1 3.040.84 10 nM AD-738033.1 2.71 0.98 10 nM AD-738034.1 4.98 3.47 10 nMAD-738035.1 1.51 0.77 10 nM AD-738036.1 1.18 1.21 10 nM AD-738037.1 2.871.38 10 nM AD-738038.1 1.52 0.43 10 nM AD-738039.1 5.43 2.42 10 nMAD-738040.1 12.15 3.03 10 nM AD-738041.1 4.14 2.38 10 nM AD-738042.14.41 2.78 10 nM AD-738043.1 0.67 0.51 10 nM AD-738044.1 1.21 0.74 10 nMAD-738045.1 21.32 2.05 10 nM AD-738046.1 8.41 3.63 10 nM AD-738047.11.92 1.96 10 nM AD-738048.1 0.83 0.24 10 nM AD-397217.2 14.29 4.68 10 nMAD-738049.1 4.40 2.05 10 nM AD-738050.1 1.46 0.17 10 nM AD-738051.1 1.481.43 10 nM AD-738052.1 4.60 0.68 10 nM AD-738053.1 3.92 1.90 10 nMAD-738054.1 6.95 1.84 10 nM AD-738055.1 2.82 0.53 10 nM AD-738056.1 4.833.07 10 nM AD-738057.1 4.79 3.01 10 nM AD-738058.1 12.43 4.84 10 nMAD-738059.1 5.66 1.40 10 nM AD-738060.1 4.24 0.94 10 nM AD-738061.110.85 2.10 10 nM AD-738062.1 1.34 0.51 10 nM AD-738063.1 31.40 6.43 10nM AD-738064.1 0.77 0.71 10 nM AD-738065.1 6.43 1.80 10 nM AD-738066.130.73 12.64 10 nM AD-738067.1 3.79 0.76 10 nM AD-738068.1 4.60 1.19 10nM AD-738069.1 36.14 12.51 10 nM AD-738070.1 34.99 13.86 10 nMAD-738071.1 1.84 1.71 10 nM AD-738072.1 1.29 1.22 10 nM AD-738073.1 0.650.14 10 nM AD-738074.1 1.28 0.51 10 nM AD-738075.1 75.00 22.72 10 nMAD-738076.1 19.31 2.56 10 nM AD-738077.1 5.21 1.66 10 nM AD-738078.17.24 5.26 10 nM AD-738079.1 1.64 0.72 10 nM AD-738080.1 2.17 1.31 10 nMAD-738081.1 13.03 2.64 10 nM AD-738082.1 3.37 1.05 10 nM AD-738083.15.36 2.87 10 nM AD-738084.1 22.04 7.85 10 nM AD-738085.1 6.81 1.80 10 nMAD-738086.1 35.05 12.18 10 nM AD-738087.1 0.14 0.10 10 nM AD-738088.134.43 18.92 10 nM AD-738089.1 11.16 1.48 10 nM AD-738090.1 4.55 0.77 10nM AD-738091.1 9.04 2.02 10 nM AD-738092.1 48.12 5.51 10 nM AD-738093.147.41 11.32 10 nM AD-738094.1 25.25 3.17 10 nM AD-738095.1 8.80 1.79 10nM AD-738096.1 4.36 5.22 10 nM AD-738097.1 28.80 6.91 10 nM AD-738098.110.91 3.70 10 nM AD-738099.1 25.30 5.42 10 nM AD-738100.1 43.27 10.46 10nM AD-738012.1 3.70 3.79 1 nM AD-738013.1 6.87 3.98 1 nM AD-738014.116.20 4.78 1 nM AD-738015.1 15.97 3.04 1 nM AD-738016.1 11.33 4.08 1 nMAD-738017.1 3.91 2.43 1 nM AD-738018.1 9.79 5.33 1 nM AD-738019.1 5.904.65 1 nM AD-738020.1 4.29 7.33 1 nM AD-738021.1 11.55 7.48 1 nMAD-738022.1 12.06 4.21 1 nM AD-738023.1 10.50 4.50 1 nM AD-738024.112.71 3.60 1 nM AD-738025.1 42.61 8.91 1 nM AD-738026.1 7.13 2.81 1 nMAD-738027.1 1.14 0.44 1 nM AD-738028.1 2.99 4.01 1 nM AD-738029.1 8.814.91 1 nM AD-738030.1 15.88 4.68 1 nM AD-738031.1 14.42 9.04 1 nMAD-738032.1 12.11 3.28 1 nM AD-738033.1 17.47 13.61 1 nM AD-738034.118.58 6.98 1 nM AD-738035.1 7.64 6.58 1 nM AD-738036.1 2.84 2.90 1 nMAD-738037.1 11.17 3.62 1 nM AD-738038.1 10.23 4.82 1 nM AD-738039.1 9.612.76 1 nM AD-738040.1 54.47 14.10 1 nM AD-738041.1 15.86 6.31 1 nMAD-738042.1 15.96 6.61 1 nM AD-738043.1 2.26 2.61 1 nM AD-738044.1 4.544.76 1 nM AD-738045.1 25.51 7.28 1 nM AD-738046.1 30.32 10.02 1 nMAD-738047.1 16.25 7.68 1 nM AD-738048.1 9.07 3.25 1 nM AD-397217.2 48.1612.70 1 nM AD-738049.1 7.97 3.33 1 nM AD-738050.1 5.60 4.81 1 nMAD-738051.1 1.49 1.05 1 nM AD-738052.1 10.13 2.72 1 nM AD-738053.1 10.824.44 1 nM AD-738054.1 21.52 8.71 1 nM AD-738055.1 12.40 3.31 1 nMAD-738056.1 5.93 4.14 1 nM AD-738057.1 7.63 2.80 1 nM AD-738058.1 18.214.26 1 nM AD-738059.1 14.39 6.00 1 nM AD-738060.1 6.71 2.99 1 nMAD-738061.1 13.63 3.65 1 nM AD-738062.1 6.08 3.37 1 nM AD-738063.1 9.638.05 1 nM AD-738064.1 6.51 4.83 1 nM AD-738065.1 9.97 1.82 1 nMAD-738066.1 50.95 5.44 1 nM AD-738067.1 9.69 2.74 1 nM AD-738068.1 9.391.51 1 nM AD-738069.1 43.67 8.07 1 nM AD-738070.1 37.85 4.96 1 nMAD-738071.1 2.81 2.93 1 nM AD-738072.1 10.65 9.82 1 nM AD-738073.1 5.642.45 1 nM AD-738074.1 10.00 4.11 1 nM AD-738075.1 78.16 11.76 1 nMAD-738076.1 44.11 8.21 1 nM AD-738077.1 11.42 0.98 1 nM AD-738078.1 7.651.23 1 nM AD-738079.1 1.78 2.66 1 nM AD-738080.1 7.03 8.36 1 nMAD-738081.1 27.43 6.11 1 nM AD-738082.1 21.57 4.04 1 nM AD-738083.110.77 3.72 1 nM AD-738084.1 76.60 10.91 1 nM AD-738085.1 36.65 7.82 1 nMAD-738086.1 26.34 11.70 1 nM AD-738087.1 0.56 0.52 1 nM AD-738088.152.50 10.17 1 nM AD-738089.1 12.77 1.25 1 nM AD-738090.1 12.92 5.28 1 nMAD-738091.1 20.70 1.73 1 nM AD-738092.1 58.85 6.24 1 nM AD-738093.184.82 9.95 1 nM AD-738094.1 59.17 6.38 1 nM AD-738095.1 12.86 8.99 1 nMAD-738096.1 10.61 4.77 1 nM AD-738097.1 35.98 1.81 1 nM AD-738098.114.76 3.12 1 nM AD-738099.1 37.99 2.57 1 nM AD-738100.1 46.62 7.08 1 nMAD-738012.1 11.95 6.41 0.1 nM AD-738013.1 11.70 2.86 0.1 nM AD-738014.133.48 9.61 0.1 nM AD-738015.1 25.02 5.00 0.1 nM AD-738016.1 22.29 4.670.1 nM AD-738017.1 21.12 5.92 0.1 nM AD-738018.1 15.82 5.90 0.1 nMAD-738019.1 22.54 18.17 0.1 nM AD-738020.1 12.05 9.08 0.1 nM AD-738021.119.21 0.85 0.1 nM AD-738022.1 24.55 5.38 0.1 nM AD-738023.1 17.43 5.050.1 nM AD-738024.1 24.48 1.96 0.1 nM AD-738025.1 72.34 16.04 0.1 nMAD-738026.1 44.09 2.91 0.1 nM AD-738027.1 16.46 9.70 0.1 nM AD-738028.113.92 9.68 0.1 nM AD-738029.1 25.75 5.87 0.1 nM AD-738030.1 42.80 8.110.1 nM AD-738031.1 43.85 2.58 0.1 nM AD-738032.1 29.64 6.11 0.1 nMAD-738033.1 42.40 1.69 0.1 nM AD-738034.1 49.71 3.53 0.1 nM AD-738035.130.30 20.42 0.1 nM AD-738036.1 12.98 4.90 0.1 nM AD-738037.1 13.01 5.340.1 nM AD-738038.1 15.19 8.17 0.1 nM AD-738039.1 18.24 10.33 0.1 nMAD-738040.1 60.24 13.10 0.1 nM AD-738041.1 26.49 7.47 0.1 nM AD-738042.118.54 6.11 0.1 nM AD-738043.1 5.91 5.08 0.1 nM AD-738044.1 14.74 6.150.1 nM AD-738045.1 55.58 16.72 0.1 nM AD-738046.1 68.30 11.74 0.1 nMAD-738047.1 40.80 6.70 0.1 nM AD-738048.1 32.28 7.47 0.1 nM AD-397217.276.28 11.27 0.1 nM AD-738049.1 22.10 8.60 0.1 nM AD-738050.1 8.56 5.260.1 nM AD-738051.1 19.62 9.00 0.1 nM AD-738052.1 29.60 6.17 0.1 nMAD-738053.1 19.82 6.73 0.1 nM AD-738054.1 48.02 6.33 0.1 nM AD-738055.126.00 8.90 0.1 nM AD-738056.1 34.85 7.55 0.1 nM AD-738057.1 30.60 9.350.1 nM AD-738058.1 49.45 11.76 0.1 nM AD-738059.1 40.24 4.74 0.1 nMAD-738060.1 37.94 10.19 0.1 nM AD-738061.1 49.79 3.08 0.1 nM AD-738062.128.19 1.51 0.1 nM AD-738063.1 30.80 15.24 0.1 nM AD-738064.1 25.32 2.670.1 nM AD-738065.1 34.43 9.76 0.1 nM AD-738066.1 87.77 14.39 0.1 nMAD-738067.1 36.47 9.15 0.1 nM AD-738068.1 28.08 4.14 0.1 nM AD-738069.197.43 7.31 0.1 nM AD-738070.1 82.37 8.24 0.1 nM AD-738071.1 27.61 7.940.1 nM AD-738072.1 37.34 2.31 0.1 nM AD-738073.1 25.85 9.17 0.1 nMAD-738074.1 41.19 13.50 0.1 nM AD-738075.1 93.48 11.50 0.1 nMAD-738076.1 66.05 10.10 0.1 nM AD-738077.1 32.71 5.69 0.1 nM AD-738078.135.64 5.42 0.1 nM AD-738079.1 20.48 3.52 0.1 nM AD-738080.1 36.41 7.720.1 nM AD-738081.1 65.34 19.91 0.1 nM AD-738082.1 53.82 8.31 0.1 nMAD-738083.1 30.04 5.11 0.1 nM AD-738084.1 88.32 9.40 0.1 nM AD-738085.178.53 7.08 0.1 nM AD-738086.1 82.59 7.90 0.1 nM AD-738087.1 13.94 6.270.1 nM AD-738088.1 73.47 18.72 0.1 nM AD-738089.1 48.21 8.12 0.1 nMAD-738090.1 43.23 12.93 0.1 nM AD-738091.1 52.45 4.67 0.1 nM AD-738092.175.75 31.47 0.1 nM AD-738093.1 88.99 10.31 0.1 nM AD-738094.1 82.41 6.940.1 nM AD-738095.1 51.05 7.29 0.1 nM AD-738096.1 31.49 12.31 0.1 nMAD-738097.1 64.39 13.12 0.1 nM AD-738098.1 33.73 10.09 0.1 nMAD-738099.1 69.09 9.27 0.1 nM AD-738100.1 75.77 15.74 0.1 nM

Example 6. In Vivo APP Screening of Sequences with AU-Rich Seeds

In vivo screening was performed on C57BL/6 mice to test oligonucleotideshaving AU-rich seeds. A summary of the study design is presented inTable 19. As shown in Table 20A, the following oligonucleotides havingAU-rich seeds were tested: AD-506935.2, AD-507065.2, AD-507159.2,AD-507538.2, AD-507624.2, AD-507724.2, AD-507725.2, AD-507789.2,AD-507874.2, AD-507928.2, and AD-507949.2. Table 20A enumerates thesense, antisense, and target oligonucleotide sequences for each of theseAU-rich oligonucleotides. The oligonucleotide AD-392927.2 (GNAT C16chemistry) from RLD592 was used as a positive control sequence. Thestructures of the AU-rich oligonucleotides are shown in FIGS. 14A and14B. Additionally, each of the oligonucleotides having AU-rich seeds wastested for cross-reactivity in human (e.g., assayed via the NM_000484sequence), cynomolgous monkey (assayed via the XM_005548883 sequence),mouse (assayed via the NM_001198823 sequence), rat (assayed via theNM_019288 sequence), and dog (assayed via the NM_001293279 sequence),and this data is summarized in Table 20B.

TABLE 19 Study Design Overview In vitro rep 646 APP NM_201414.2 TargethsAPP Goal In vivo screen of sequences with AU-rich seeds AAV NameAAV8.HsAPP-CDS3TRNC VCAV-04731 Dose 2E+11 Injection method IV (retroorbital) siRNA Injection method Subcutaneous Dose 3 mg/kg Sample LiverCollection days D 14 Animals Sex Female Strain C57BL/6 Age (on arrival)6-8 weeks Vendor Charles River Duplex No. 12* n=  3 Total animals 45Analysis Analysis method RT-qPCR Taqman probe APP: Hs00169098_m1 (FAM)Mouse GAPDH Applied Biosystems 4351309 (VIC) Misc. Controls *AD-392927.2from RLD592 was included as positive control

TABLE 20hsAPP Duplex and Target Sequences for GNA7 C16 control and AU-rich Candidates.SEQ SEQ mRNA target  SEQ Chemistry Duplex ID Antisense Sequence  IDsequence  ID (Target) Name Sense Sequence (5′ to 3′) NO (5′ to 3′) NO(5′ to 3′) NO GNA7 AD- usasgug(Chd)AfuGfAfAfuaga 2750asGfsagaa(Tgn)cuauu 2751 n/a n/a C16 392927.2 uucucuL96 cAfuGfcacuasgsu(APP) AU-rich AD- asasagagCfaAfAfAfcuauucag 2752 asUfscugAfaUfAfguuu2753 AGAAAGAGCAAA 2754 seed 506935.2 auL96 UfgCfucuuuscsu ACUAUUCAGAU(APP) AU-rich AD- ususggccAfaCfAfUfgauuagug 2755 asUfscacUfaAfUfcaug2756 UCUUGGCCAACA 2757 seed 507065.2 auL96 UfuGfgccaasgsa UGAUUAGUGAA(APP) AU-rich AD- uscsugggUfuGfAfCfaaauauca 2758 asUfsugaUfaUfUfuguc2759 GUUCUGGGUUGA 2760 seed 507159.2 auL96 AfaCfccagasasc CAAAUAUCAAG(APP) AU-rich AD- ususuaugAfnUfUfAfcucauuau 2761 asGfsauaAfuGfAfguaa2762 GUUUUAUGAUUU 2763 seed 507538.2 cuL96 AfuCfauaaasasc ACUCAUUAUCG(APP) AU-rich AD- asusgccuGfaAfCfUfugaauuaa 2764 asAfsuuaAfnUfCfaagu2765 AGAUGCCUGAAC 2766 seed 507624.2 uuL96 UfcAfggcauscsu UUGAAUUAAUC(APP) AU-rich AD- asgsaugcCfuGfAfAfcuugaauu 2767 asUfsaauUfcAfAfguuc2768 GUAGAUGCCUGA 2769 seed 507724.2 auL96 AfgGfcaucusasc ACUUGAAUUAA(APP) AU-rich AD- gscscugaAfcUfUfGfaauuaauc 2770 asGfsgauUfaAfUfucaa2771 AUGCCUGAACUU 2772 seed 507725.2 cuL96 GfuUfcaggcsasu GAAUUAAUCCA(APP) AU-rich AD- gsusgguuUfgUfGfAfcccaauua 2773 asUfsuaaUfuGfGfguca2774 UUGUGGUUUGUG 2775 seed 507789.2 auL96 CfaAfaccacsasa ACCCAAUUAAG(APP) AU-rich AD- csasgaugCfuUfUfAfgagagauu 2776 asAfsaauCfuCfUfcuaa2777 UUCAGAUGCUUU 2778 seed 507874.2 uuL96 AfgCfaucugsasa AGAGAGAUUUU(APP) AU-rich AD- uscsuugcCfuAfAfGfuauuccuu 2779 asAfsaagGfaAfUfacuu2780 UCUCUUGCCUAA 2781 seed 507928.2 uuL96 AfgGfcaagasgsa GUAUUCCUUUC(APP) AU-rich AD- ususgcugCfuUTCfUfgcuauauu 2782 asAfsaauAfuAfGfcaga2783 GAUUGCUGCUUC 2784 seed 507949.2 uuL96 AfgCfagcaasusc UGCUAUAUUUG(APP) Table 20 key: U = uridine-3′-phosphate, u= 2′-O-methyluridine-3′-phosphate, us= 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf= 2′-fluoroguanosine-3′-phosphate, Uf = 2′-fluorouridine-3′-phosphate,Cf = 2′-fluorocytidine-3′-phosphate, Af= 2′-fluoroadenosine-3′-phosphate, cs = 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn) = Adenosine-glycol nucleic acid (GNA),gs = 2′-O-methylguanosine-3′-phosphorothioate, (Chd)= 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) = 2′-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate.

Selected AU-rich candidates were evaluated for in vivo efficacy in leadidentification screens for human APP knockdown in AAV mice. Briefly, anAAV vector harboring Homo sapiens APP (e.g., hsAPP-CDS3TRNC) wasintravenously injected into 6-8 week old C57BL/6 female mice, and at 14days post-AAV administration a selected RNAi agent or a control agentwas subcutaneously injected into the mice (n=3 per group at a dose of 3mg/kg. The screening groups are summarized in Table 21.

TABLE 21 Screening Groups for AU-rich Candidates in AAV Mice. siRNA DateGroup # Animal # Treatment Dose Timepoint 8 Mar. 2019 1 1 PBS N/A D 14 8Mar. 2019 1 2 PBS N/A D 14 8 Mar. 2019 1 3 PBS N/A D 14 8 Mar. 2019 1 4PBS N/A D 14 8 Mar. 2019 1 5 PBS N/A D 14 8 Mar. 2019 2 6 Naïve N/A D 148 Mar. 2019 2 7 Naïve N/A D 14 8 Mar. 2019 2 8 Naïve N/A D 14 8 Mar.2019 2 9 Naïve N/A D 14 8 Mar. 2019 3 10 AD-392927.2 3 mg/kg D 14 (fromRLD592) 8 Mar. 2019 3 11 AD-392927.2 3 mg/kg D 14 (from RLD592) 8 Mar.2019 3 12 AD-392927.2 3 mg/kg D 14 (from RLD592) 8 Mar. 2019 4 13AD-506935.2 3 mg/kg D 14 8 Mar. 2019 4 14 AD-506935.2 3 mg/kg D 14 8Mar. 2019 4 15 AD-506935.2 3 mg/kg D 14 8 Mar. 2019 5 16 AD-507065.2 3mg/kg D 14 8 Mar. 2019 5 17 AD-507065.2 3 mg/kg D 14 8 Mar. 2019 5 18AD-507065.2 3 mg/kg D 14 8 Mar. 2019 6 19 AD-507159.2 3 mg/kg D 14 8Mar. 2019 6 20 AD-507159.2 3 mg/kg D 14 8 Mar. 2019 6 21 AD-507159.2 3mg/kg D 14 8 Mar. 2019 7 22 AD-507538.2 3 mg/kg D 14 8 Mar. 2019 7 23AD-507538.2 3 mg/kg D 14 8 Mar. 2019 7 24 AD-507538.2 3 mg/kg D 14 8Mar. 2019 8 25 AD-507624.2 3 mg/kg D 14 8 Mar. 2019 8 26 AD-507624.2 3mg/kg D 14 8 Mar. 2019 8 27 AD-507624.2 3 mg/kg D 14 8 Mar. 2019 9 28AD-507724.2 3 mg/kg D 14 8 Mar. 2019 9 29 AD-507724.2 3 mg/kg D 14 8Mar. 2019 9 30 AD-507724.2 3 mg/kg D 14 8 Mar. 2019 10 31 AD-507725.2 3mg/kg D 14 8 Mar. 2019 10 32 AD-507725.2 3 mg/kg D 14 8 Mar. 2019 10 33AD-507725.2 3 mg/kg D 14 8 Mar. 2019 11 34 AD-507789.2 3 mg/kg D 14 8Mar. 2019 11 35 AD-507789.2 3 mg/kg D 14 8 Mar. 2019 11 36 AD-507789.2 3mg/kg D 14 8 Mar. 2019 12 37 AD-507874.2 3 mg/kg D 14 8 Mar. 2019 12 38AD-507874.2 3 mg/kg D 14 8 Mar. 2019 12 39 AD-507874.2 3 mg/kg D 14 8Mar. 2019 13 40 AD-507928.2 3 mg/kg D 14 8 Mar. 2019 13 41 AD-507928.2 3mg/kg D 14 8 Mar. 2019 13 42 AD-507928.2 3 mg/kg D 14 8 Mar. 2019 14 43AD-507949.2 3 mg/kg D 14 8 Mar. 2019 14 44 AD-507949.2 3 mg/kg D 14 8Mar. 2019 14 45 AD-507949.2 3 mg/kg D 14

The mice were sacrificed and their livers were assessed for APP mRNAlevels at 14 days post-subcutaneous injection of RNAi agent or controlby qPCR. As shown in Table 22 and FIG. 15, significant levels of in vivohuman APP mRNA knockdown in liver were observed for most AU-rich RNAiagents tested, as compared to PBS and Naïve (AAV only) controls, withparticularly robust levels of knockdown observed for AD-507538.2,AD-507724.2, AD-392927.2 (RLD592), AD-507928.2, AD-506935.2, andAD-507874.2

TABLE 22 Summary of Screening Results for AU-rich Candidates in AAVMice. hsAPP AU rich seed 3 mg/kg liver qPCR D 14 % hsAPP messageremaining relative to PBS Treatment Group Average Standard Deviation PBS100.00 30.90 Naïve 91.48 6.43 AD-507538.2 24.23 8.73 AD-507724.2 30.902.95 AD-392927.2 (RLD592) 32.80 0.92 AD-507928.2 36.31 2.61 AD-506935.236.47 5.26 AD-507874.2 40.43 4.99 AD-507789.2 50.24 6.06 AD-507624.257.67 4.22 AD-507725.2 68.53 10.04 AD-507159.2 71.49 20.82 AD-507949.284.94 2.35 AD-507065.2 91.09 20.17

Table 23 shows a comparison of in vivo human hsAPP mRNA knockdown inliver by the above-described AU-rich RNAi agents at 3 mg/kg as comparedto in vitro APP knockdown of the same AU-rich RNAi agents at either 10nM or 0.1 nM in both DL and Be(2)C cell lines.

TABLE 23 Comparison of In Vivo vs. In Vitro hsAPP Knockdown. RLD 646 (invitro) DL (dual-Luc) Be(2)C (human neuron) hsAPP % RLD 701 Dose -- UnitDose -- Unit Dose -- Unit Dose -- Unit remaining In vivo 3 mg/kg 10 nM10 nM 0.1 nM 0.1 nM 10 nM 10 nM 0.1 nM 0.1 nM Duplex Avg SD Avg SD AvgSD Avg SD Avg SD AD-507538 24.2 8.7 22.5 5.5 106.2 45.3 16.6 3.8 23.32.5 AD-507724 30.9 2.9 38.5 9.2 119.8 24.6 21.2 1.9 43.5 9.6 AD-50792836.3 2.6 10.6 1.8 101.1 23.8 25.1 2.7 39.8 22.1 AD-506935 36.5 5.3 37.410.1 75.9 18.4 19.7 3.9 22.3 2.2 AD-507874 40.4 5.0 13.6 10.9 105.7 29.121.9 2.4 31.3 13.1 AD-507789 50.2 6.1 34.3 12.0 121.2 30.9 24.0 6.0 38.33.6 AD-507624 57.7 4.2 32.7 7.0 116.5 28.6 22.1 1.0 68.0 26.2 AD-50772568.5 10.0 68.6 13.1 107.8 43.5 31.1 5.4 34.1 9.7 AD-507159 71.5 20.856.8 20.2 119.7 42.4 26.2 4.6 42.7 8.6 AD-507949 84.9 2.3 57.1 25.6 99.729.8 23.3 3.1 42.9 15.2 AD-507065 91.1 20.2 52.5 11.6 106.1 19.2 25.37.2 39.4 5.8

Example 7. In Vivo APP Screening of Lead Sequences for StructureActivity Relationship Studies

In vivo screening was performed on C57BL/6 mice to conduct structureactivity relationship studies on lead oligonucleotides. A summary of thestudy design is presented in Table 24. As shown in Table 25, thefollowing lead oligonucleotides were tested: AD-886823, AD-886839,AD-886845, AD-886853, AD-886858, AD-886864, AD-886873, AD-886877,AD-886879, AD-886883, AD-886884, AD-886889, AD-886899, AD-886900,AD-886906, AD-886907, AD-886908, AD-886909, AD-886919, AD-886928,AD-886930 and AD-886931. Table 25 lists sense and antisense sequencesfor each lead oligonucleotide, as well as the associated target sequencefor each lead oligonucleotide. The structures of the leadoligonucleotides are shown in FIG. 16A, FIG. 16B, FIG. 16C, and FIG.16D.

TABLE 24 Study Design AAV Name AAV8.HsAPP-CDS3TRNC VCAV-04731 Dose 2E+11Injection method IV (retro orbital) siRNA Injection method SubcutaneousDose 3 mg/kg Sample Liver Collection days D 14 Animals Sex Female StrainC57BL/6 Age (on arrival) 6-8 weeks Vendor Jackson Lab Duplex No. 16 n= 3 Total animals 72 Analysis Analysis method RT-qPCR Taqman probe MouseGAPDH Applied Biosystems 4351309 APP: Hs00169098_m1 (FAM)

TABLE 25 hsAPP Duplex and Target Sequences for SAR Lead Candidates. SEQSEQ ID ID Duplex Strand Oligonucleotide Sequence NO: Target Sequence NO:AD- Sense usasgug(Chd)AfuGfAfAfuag 2785 UAGUGCAUGAAUAGAUUCUCA 2829886823.2 (5′ to 3′) auucucaL96 Antisense VPusGfsagaa(Tgn)cuauucAf 2786UGAGAATCUAUUCAUGCACUAGU 2830 (5′ to 3′) uGfcacuasgsu AD- Senseusasgug(Chd)AfudGAfAfuag 2787 UAGUGCAUGAAUAGAUUCUCA 2831 886839.2(5′ to 3′) auucucaL96 Antisense VPusdGsagaa(Tgn)cuauucAf 2788UGAGAATCUAUUCAUGCACUAGU 2832 (5′ to 3′) udGcacuasgsu AD- Senseusasgug(Chd)audGadAuagau 2789 UAGUGCAUGAAUAGAUUCUCA 2833 886845.2(5′ to 3′) ucucaL96 Antisense VPudGagaa(Tgn)cuauUfcAfu 2790UGAGAATCUAUUCAUGCACUAGU 2834 (5′ to 3′) dGcacuasgsu AD- Senseusasgug(Chd)AfudGAfAfuag 2791 UAGUGCAUGAAUAGAUUCUCA 2835 886853.2(5′ to 3′) auucucaL96 Antisense VPusdGsagdAa(Tgn)cuaudTc 2792UGAGAATCUAUTCAUGCACUAGU 2836 (5′ to 3′) Afugcacuasgsu AD- Senseusasgug(Chd)AfudGAfAfuag 2793 UAGUGCAUGAAUAGAUUCUCA 2837 886858.2(5′ to 3′) auucucaL96 Antisense VPudGagdAa(Tgn)cuaudTcAf 2794UGAGAATCUAUTCAUGCACUAGU 2838 (5′ to 3′) udGcacuasgsu AD- Sensegsgscua(Chd)GfaAfAfAfucc 2795 GGCUACGAAAAUCCAACCUAA 2839 886864.2(5′ to 3′) aaccuaaL96 Antisense VPusUfsaggu(Tgn)ggauuuUf 2796UUAGGUTGGAUUUUCGUAGCCGU 2840 (5′ to 3′) cGfuagccsgsu AD- Sensegsgscua(Chd)gadAadAuccaa 2797 GGCUACGAAAAUCCAACCUAA 2841 886873.2(5′ to 3′) ccuaaT96 Antisense VPusUfsaggu(Tgn)ggandTnU 2798UUAGGUTGGAUTUUCGUAGCCGU 2842 (5′ to 3′) fcdGuagccsgsn AD- Sensegsgscua(Chd)gadAadAuccaa 2799 GGCUACGAAAAUCCAACCUAA 2843 886877.2(5′ to 3′) ccuanT96 Antisense VPusUfsaggu(Tgn)ggandTnU 2800UUAGGUTGGAUTUUCGUAGCCGU 2844 (5′ to 3′) fcguagccsgsn AD- Sensegsgscua(Chd)gaAfAfAfucca 2801 GGCUACGAAAAUCCAACCUAA 2845 886879.2(5′ to 3′) accuaaL96 Antisense VPusUfsaggu(Tgn)gganunUf 2802UUAGGUTGGAUUUUCGUAGCCGU 2846 (5′ to 3′) cdGuagccsgsn AD- Sensegsgscua(Chd)gaAfAfAfucca 2803 GGCUACGAAAAUCCAACCUAA 2847 886883.2(5′ to 3′) accuaaL96 Antisense VPuUfaggdT(Tgn)gganunUfcd 2804UUAGGTTGGAUUUUCGUAGCCGU 2848 (5′ to 3′) Guagccsgsn AD- Sensegsgscua(Chd)gaAfAfAfuccaa 2805 GGCUACGAAAAUCCAACCUAA 2849 886884.2(5′ to 3′) ccuaaL96 Antisense VPuUfaggdT(Tgn)ggandTnUfc 2806UUAGGTTGGAUTUUCGUAGCCGU 2850 (5′ to 3′) dGuagccsgsn AD- Sensegsgscua(Chd)gaAfAfAfuccaa 2807 GGCUACGAAAAUCCAACCUAA 2851 886889.2(5′ to 3′) ccuaaL96 Antisense VPuUfagdGn(Tgn)ggandTuUfc 2808UUAGGUTGGAUTUUCGUAGCCGU 2852 (5′ to 3′) guagccsgsn AD- SenseasasagagCfaAfAfAfcuann 2809 AAAGAGCAAAACUAUUCAGAA 2853 886899.2(5′ to 3′) (Chd)aganT96 Antisense VPusUfscugAfaUfAfgunnUfgC 2810UUCUGAAUAGUUUUGCUCUUUCU 2854 (5′ to 3′) fncuunscsu AD- Senseasasaga(Ghd)CfaAfAfAfcuan 2811 AAAGAGCAAAACUAUUCAGAA 2855 886900.2(5′ to 3′) ucaganT96 Antisense VPusUfscugAfauaguuuUfgCfu 2812UUCUGAAUAGUUUUGCUCUUUCU 2856 (5′ to 3′) cuuuscsu AD- Senseasasagag(Chd)aAfAfAfcuauu 2813 AAAGAGCAAAACUAUUCAGAA 2857 886906.2(5′ to 3′) cagaaL96 Antisense VPuUfcugAfauaguunUfgCfucu 2814UUCUGAAUAGUUUUGCUCUUUCU 2858 (5′ to 3′) uuscsu AD- Senseasasagag(Chd)aAfaAfcuauuc 2815 AAAGAGCAAAACUAUUCAGAA 2859 886907.2(5′ to 3′) agaaL96 Antisense VPuUfcugAfauagudTuUfgCfuc 2816UUCUGAAUAGUTUUGCUCUUUCU 2860 (5′ to 3′) uuuscsu AD- Senseasasagag(Chd)adAadAcuauuc 2817 AAAGAGCAAAACUAUUCAGAA 2861 886908.2(5′ to 3′) agapT96 Antisense VPuUfcugAfauagudTuUfgCfuc 2818UUCUGAAUAGUTUUGCUCUUUCU 2862 (5′ to 3′) uuuscsu AD- Senseasasagag(Chd)adAadAcuauuc 2819 AAAGAGCAAAACUAUUCAGAA 2863 886909.2(5′ to 3′) agapT96 Antisense VPuUfcugdAauagudTuUfgdCuc 2820UUCUGAAUAGUTUUGCUCUUUCU 2864 (5′ to 3′) uuuscsu AD- Senseususuau(Ghd)AftfUTUfAfcuc 2821 UUUAUGAUUUACUCAUUAUCA 2865 886919.2(5′ to 3′) auuaucaL96 Antisense VPusGfsauaAfugaguaaAfuCfa 2822UGAUAAUGAGUAAAUCAUAAAAC 2866 (5′ to 3′) uaaasasc AD- Senseususuaug(Ahd)uUfudAcucauu 2823 UUUAUGAUUUACUCAUUAUCA 2867 886928.2(5′ to 3′) aucaL96 Antisense VPudGauadAugagudAaAfudCau 2824UGAUAAUGAGUAAAUCAUAAAAC 2868 (5′ to 3′) aaasasc AD- SenseususuaugAfuUTUfAfcuc(Ahd) 2825 UUUAUGAUUUACUCAUUAUCA 2869 886930.2(5′ to 3′) uuaucaL96 Antisense VPusdGsauaAfugaguaaAfuCfa 2826UGAUAAUGAGUAAAUCAUAAAUG 2870 (5′ to 3′) uaaasusg AD- Senseususuaug(Ahd)uUfUfAfcucau 2827 UUUAUGAUUUACUCAUUAUCA 2871 886931.2(5′ to 3′) uaucaL96 Antisense VPusdGsanaAfugaguaaAfuCfa 2828UGAUAAUGAGUAAAUCAUAAAUG 2872 (5′ to 3′) uaaasusg Table 25 key: U= uridine-3-phosphate, u = 2′-O-methyluridine-3′-phosphate, us= 2′-O-methyluridine-3′-phosphorothioate, a= 2′-O-methyladenosine-3′-phosphate, A = adenosine-3′-phosphate, as= 2′-O-methyladenosine-3′-phosphorothioate, (Ahd)= 2′-O-hexadecyl-adenosine-3′-phosphate, Gf= 2′-fluoroguanosine-3′-phosphate, Uf = 2′-fluorouridine-3′-phosphate,Cf = 2′-fluorocytidine-3′-phosphate, Af= 2′-fluoroadenosine-3′-phosphate, cs= 2′-O-methylcytidine-3′-phosphate, VP = Vinylphosphate 5′, (Agn)= Adenosine-glycol nucleic acid (GNA), gs= 2′-O-methylguanosine-3′-phosphorothioate, (Chd)= 2′-O-hexadecyl-cytidine-3′-phosphate, (Tgn) = Thymidine-glycol nucleicacid (GNA) S-Isomer, (Ghd) = 21-O-hexadecyl-guanosine-3′-phosphate, andcs = 2′-O-methylcytidine-3′-phosphorothioate. Selected candidates wereevaluated for in vivo efficacy in screens for human APP knockdown in AAVmice. Briefly, an AAV vector harboring Homo sapiens APP (e.g.,AAV8.HsAPP-CDS3TRNC) was intravenously injected into 6-8 week oldC57BL/6 female mice, and at 14 days post-AAV administration a selectedRNAi agent or a control agent was subcutaneously injected into the mice(n/per group) at a dose of 3 mg/kg. The mice were sacrificed and theirlivers were assessed for APP mRNA levels at 14 days post-subcutaneousinjection of RNAi agent or control by qPCR. As shown in Table 26, FIG.17A, and FIG. 17B, significant levels of in vivo human APP mRNAknockdown in liver were observed for most lead RNAi agents tested, ascompared to PBS and Naïve (AAV only) controls, with particularly robustlevels of knockdown observed for AD-886864 (parent), AD-886873,AD-886879, AD-886883, AD-886884, AD-886889, AD-886899 (parent),AD-886900, AD-886906, AD-886907, AD-886919 (parent), and AD-886823(parent) FIGS. 18A-18D are schematic representations of the lead RNAiagents classified by parent molecule: AD-886864 parent (FIG. 18A),AD-886899 parent (FIG. 18B), AD-886919 parent (FIG. 18 C), and AD-886823parent (FIG. 18D), respectively

TABLE 26 Summary of In Vivo Screening Results for Lead Candidates in AAVMice. 3 mg/kg SC D14 liver qPCR % message remaining Standard TreatmentGroup Average Deviation PBS 100.00 15.26 naïve (AAV-only) 104.01 16.49AD-886864 (parent) 29.55 0.93 AD-886873 27.48 0.84 AD-886877 33.34 13.46AD-886879 26.68 2.52 AD-886883 21.74 2.25 AD-886884 28.51 8.66 AD-88688921.77 1.58 AD-886899 (parent) 27.17 7.52 AD-886900 20.80 5.81 AD-88690619.35 5.97 AD-886907 19.12 3.16 AD-886908 30.28 6.67 AD-886909 34.565.55 AD-886919 (parent) 24.16 6.71 AD-886928 40.47 5.03 AD-886930 32.876.63 AD-886931 38.82 4.51 AD-886823 (parent) 27.81 3.36 AD-886853 43.599.18 AD-886858 61.16 2.23 AD-886839 60.35 13.85 AD-886845 79.73 10.09

Table 29 shows in vitro APP knockdown of the above-described (e.g.,Table 26) lead RNAi agents at either 10 nM or 0.1 nM in Be(2)C celllines.

TABLE 29 Summary of In Vitro Screening Results for Lead Candidates inBe(2)C Cells at 10 nM and 0.1 nM Doses. % of Message % of MessageRemaining - STD EV - Remaining - STD EV - Duplex 10 nM 10 nM 0.1 nM 0.1nM AD-886823.1 7.0 5.4 91.0 25.7 AD-886845.1 13.3 3.2 67.4 7.9AD-886839.2 10.2 7.7 74.7 39.5 AD-886853.1 6.5 3.9 44.9 7.4 AD-886858.111.4 2.4 61.4 12.5 AD-886864.1 11.5 3.9 44.9 7.9 AD-886873.1 12.7 1.960.2 14.3 AD-886877.1 11.9 3.0 67.8 5.9 AD-886879.1 9.8 1.7 41.0 5.2AD-886883.1 8.5 2.1 29.5 12.8 AD-886884.1 8.9 2.4 31.2 11.9 AD-886889.19.4 0.6 28.0 14.2 AD-886899.1 9.2 2.6 40.5 12.7 AD-886900.1 6.7 2.1 39.721.5 AD-886906.1 10.2 3.0 39.7 9.9 AD-886907.1 9.8 2.7 30.3 1.9AD-886908.1 10.7 2.6 32.8 10.6 AD-886909.1 7.4 1.4 77.9 16.2 AD-886919.15.7 1.4 31.2 4.3 AD-886928.1 9.2 2.4 67.6 7.1 AD-886930.1 6.9 1.7 45.710.1 AD-886931.1 3.2 1.2 42.0 16.0

Example 8. In Vivo Knock Down of APP Via C-16 siRNA Conjugates inNon-Human Primates

Because of the efficacy of the siRNA conjugate AD-454844, structureactivity relationship studies were carried out on AD-454844, and 5 newC16 compounds were then identified as lead compounds based on Cynomonkey in vivo screens of soluble APP. In vivo knock down effects of C16siRNA conjugates were assessed in Cyno monkeys given 60 mg of AD-454844,AD-994379, AD-961583, AD-961584, AD-961585, or AD-961586 via intrathecaladministration between L2/L3 or L4/L5 via percutaneous needle stick inthe lumbar cistern (FIGS. 20A-20G). Soluble APP alpha and beta targetengagement biomarkers were assessed from CSF collected at D8, D15 andD29 post dose. IT dosing resulted in sufficient siRNA deliverythroughout the spine and brain as demonstrated by silencing of targetengagement biomarkers as early as one week post dose with sustainedactivity through D29. Notably, the in vivo knock down activity of the 5′end C16 conjugate (AD-994379) was similar to that of the internal C16conjugate (AD-454844). (The antisense sequence is identical across bothmolecules tested).

Further, the C16 siRNA conjugates exhibited a significant long lastingknock down effect. Sustained pharmacodynamic effects in which solubleAPP remained well below 50% over a 4 month period were observedfollowing a single dose of 60 mg of AD-454844 (FIGS. 19 and 20A).

TABLE 30  C16 siRNA conjugates identified to knockdown APP in in vivo NHP studies SEQ SEQ Oligonucleotide  ID Target  IDDuplex Strand Sequence NO: Sequence NO: AD- Sense Q363sasaaaucCfaAfCf2873 CGAAAAUCCAACCUACAAGUUCU 2883 994379 (5′ to 3′) CfuacaaguuscsaAntisense VPusGfsaacu(Tgn)gua 2874 AGAACUUGUAGGUUGGAUUUUCG 2884(5′ to 3′) gguUfgGfauuuuscsg AD- Sense gsgscua(Chd)gadAadA 2875ACGGCUACGAAAAUCCAACCUAC 2885 961583 (5′ to 3′) uccaaccusasa AntisenseVPusUfsaggu(Tgn)gga 2876 GUAGGUUGGAUUUUCGUAGCCGU 2886 (5′ to 3′)udTuUfcdGuagccsgsu AD- Sense asasagag(Chd)aAfaAf 2877AGAAAGAGCAAAACUAUUCAGAU 2887 961584 (5′ to 3′) cuauucagsasa AntisenseVPuUfcugAfauagudTuU 2878 AUCUGAAUAGUUUUGCUCUUUCU 2888 (5′ to 3′)fgCfucuuuscsu AD- Sense asasagag(Chd)adAadA 2879 AGAAAGAGCAAAACUAUUCAGAU2889 961585 (5′ to 3′) cuauucagsasa Antisense VPuUfcugdAauagudTuU 2880AUCUGAAUAGUUUUGCUCUUUCU 2890 (5′ to 3′) fgdCucuuuscsu AD- Senseususuau(Ghd)AfuUfUf 2881 GUUUUAUGAUUUACUCAUUAUCG 2891 961586 (5′ to 3′)Afcucauuauscsa Antisense VPusGfsauaAfugaguaa 2882CGAUAAUGAGUAAAUCAUAAAAC 2892 (5′ to 3′) AfuCfauaaasusg

TABLE 29  Unmodified base transcripts used in theC16 conjugates of Table 30. SEQ Transcript  ID Duplex Strand Oligo nameSequence NO: AD- Sense A-1701871.1 AAAAUCCAACCU 2893 994379 (5′ to 3′)ACAAGUUCA Antisense A-882382.1 UGAACUTGUAGG 2894 (5′ to 3′) UUGGAUUUUCGAD- Sense A-1770584.1 GGCUACGAAAAU 2895 961583 (5′ to 3′) CCAACCUAAAntisense A-1683088.1 UUAGGUTGGAUT 2896 (5′ to 3′) UUCGUAGCCGU AD- SenseA-1770585.1 AAAGAGCAAAAC 2897 961584 (5′ to 3′) UAUUCAGAA AntisenseA-1683116.1 UUCUGAAUAGUT 2898 (5′ to 3′) UUGCUCUUUCU AD- SenseA-1770586.1 AAAGAGCAAAAC 2899 961585 (5′ to 3′) UAUUCAGAA AntisenseA-1683118.1 UUCUGAAUAGUT 2900 (5′ to 3′) UUGCUCUUUCU AD- SenseA-1770587.1 UUUAUGAUUUAC 2901 961586 (5′ to 3′) UCAUUAUCA AntisenseA-1683134.1 UGAUAAUGAGUA 2902 (5′ to 3′) AAUCAUAAAUG

We claim:
 1. A double stranded ribonucleic acid (RNAi) agent for inhibiting expression of an amyloid precursor protein (APP) gene, comprising: (a) a sense strand comprising the nucleotide sequence of 5′-GGCUACGAAAAUCCAACCUAA-3′ (SEQ ID NO: 2735), wherein the sense strand comprises one or more lipophilic moieties conjugated to one or more non-terminal nucleotide positions excluding positions 9-12, wherein the 5′ terminal G of SEQ ID NO: 2735 is position 1; and (b) an antisense strand comprising the nucleotide sequence of 5′-UUAGGUTGGAUTUUCGUAGCCGU-3′ (SEQ ID NO:2743); wherein the double stranded RNAi agent comprises at least one modified nucleotide.
 2. The double stranded RNAi agent of claim 1, wherein the sense strand and the antisense strand each are 30 nucleotides or less in length.
 3. The double stranded RNAi agent of claim 1, wherein a plurality of the nucleotides of the sense strand, the antisense strand, or both the sense strand and the antisense strand are modified nucleotides.
 4. The double stranded RNAi agent of claim 3, wherein substantially all of the nucleotides of the sense strand, the antisense strand, or both the sense strand and the antisense strand are modified nucleotides.
 5. The double stranded RNAi agent of claim 1, wherein the at least one modified nucleotide is selected from the group consisting of a 2′-O-methyl modification, a 2′-deoxy modification, a glycol nucleic acid (GNA), and a 2′-fluoro modification.
 6. The double stranded RNAi agent of claim 1, further comprising 6-8 phosphorothioate internucleotide linkages.
 7. The double stranded RNAi agent of claim 1, wherein at least one strand comprises a 3′ overhang of at least 2 nucleotides or the antisense strand comprises a 3′ overhang of at least 2 nucleotides.
 8. The double stranded RNAi agent of claim 1, wherein the one or more non-terminal nucleotide positions of the sense strand have the following structure:

wherein B is a nucleotide base or a nucleotide base analog.
 9. The double stranded RNAi agent of claim 1, wherein the antisense strand comprises one or more lipophilic moieties conjugated to one or more non-terminal nucleotide positions.
 10. The double stranded RNAi agent of claim 1, wherein the one or more lipophilic moieties are conjugated to any of positions 4-8 of the sense strand counting from the 5′ end of the sense strand.
 11. The double stranded RNAi agent of claim 1, further comprising a phosphate or a phosphate mimic or a 5′-vinyl phosphonate (VP) group at the 5′-end of the antisense strand.
 12. The double stranded RNAi agent of claim 1, further comprising a 5′-vinyl phosphonate (VP) group at the 5′-end of the antisense strand.
 13. The double stranded RNAi agent of claim 1, wherein the one or more lipophilic moieties is one lipophilic moiety conjugated to one nucleotide position selected from the group consisting of positions 4, 6, 7, and 8 of the sense strand counting from the 5′-end of the sense strand.
 14. The double stranded RNAi agent of claim 1, wherein the one or more lipophilic moieties are only conjugated to one or more of positions 5, 6, 7, 15, and 17 on the sense strand.
 15. The double stranded RNAi agent of claim 1, wherein the sense strand comprises the modified nucleotide sequence of 5′-gsgscua(Chd)gadAadAuccaaccusasa-3′ (SEQ ID NO:2734) and the antisense strand comprises the modified nucleotide sequence of 5′-VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu-3′ (SEQ ID NO:2742), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; s is a phosphorothioate linkage; Uf is 2′-fluoro U; dA, dG, and dT are 2′-deoxy A, G, and T; (Chd) is 2′-O-hexadecyl-cytidine-3′-phosphate; (Tgn) is Thymidine-glycol nucleic acid (GNA)S-Isomer; and VP is 5′-vinyl phosphonate.
 16. A pharmaceutical composition for inhibiting expression of an APP gene comprising the double stranded RNAi agent of claim
 15. 17. The double stranded RNAi agent of claim 1, wherein: the sense strand consists of the modified nucleotide sequence of 5′-gsgscua(Chd)gadAadAuccaaccusasa-3′ (SEQ ID NO:2734) and; the antisense strand consists of the modified nucleotide sequence of 5′-VPusUfsaggu(Tgn)ggaudTuUfcdGuagccsgsu-3′ (SEQ ID NO:2742), wherein a, g, c and u are 2′-O-methyl (2′-OMe) A, G, C, and U; s is a phosphorothioate linkage; Uf is 2′-fluoro U; dA, dG, and dT are 2′-deoxy A, G, and T; (Chd) is 2′-O-hexadecyl-cytidine-3′-phosphate; (Tgn) is Thymidine-glycol nucleic acid (GNA)S-Isomer; and VP is 5′-vinyl phosphonate.
 18. A pharmaceutical composition for inhibiting expression of an amyloid precursor protein (APP) gene comprising the double stranded RNAi agent of claim
 17. 19. A method of inhibiting expression of an amyloid precursor protein (APP) gene in a cell, the method comprising: (a) contacting the cell with the pharmaceutical composition of claim 16; and (b) maintaining the cell of step (a) in contact with the pharmaceutical composition for a time sufficient to degrade mRNA transcripts of the APP gene, thereby inhibiting expression of the APP gene in the cell.
 20. The method of claim 19, wherein the APP expression is inhibited by at least about 30%.
 21. The method of claim 19, wherein the cell is within a subject.
 22. The method of claim 21, wherein the pharmaceutical composition is administered to the subject intrathecally.
 23. The method of claim 21, wherein the subject suffers from an APP-associated disorder.
 24. The method of claim 23, wherein the APP-associated disorder is selected from the group consisting of a cerebral amyloid angiopathy (CAA), an early onset familial Alzheimer disease (EOFAD), and an Alzheimer's disease (AD).
 25. A method of inhibiting expression of an amyloid precursor protein (APP) gene in a cell, the method comprising: (a) contacting the cell with the pharmaceutical composition of claim 18; and (b) maintaining the cell of step (a) in contact with the pharmaceutical composition for a time sufficient to obtain degradation of the mRNA transcript of an APP gene, thereby inhibiting expression of the APP gene in the cell.
 26. The method of claim 25, wherein the APP expression is inhibited by at least about 30%.
 27. The method of claim 26, wherein the cell is within a subject.
 28. The method of claim 27, wherein the pharmaceutical composition is administered to the subject intrathecally.
 29. The method of claim 27, wherein the subject suffers from an APP-associated disorder.
 30. The method of claim 29, wherein the APP-associated disease is cerebral amyloid angiopathy (CAA), early onset familial Alzheimer disease (EOFAD), or Alzheimer's disease (AD). 